Trefoil family factor proteins and uses thereof

ABSTRACT

The invention provides for methods for treating an inflammatory disease of the digestive system in a subject by administering a trefoil family molecule. The invention provides for methods for treating a digestive system cancer in a subject by administering a trefoil family molecule. The invention provides for methods for cell proliferation in a subject by administering a trefoil family molecule.

This application is a continuation-in-part of International ApplicationNo. PCT/US2013/034981, filed Apr. 2, 2013, which claims priority to U.S.Provisional Patent Application No. 61/649,767, filed on May 21, 2012,the contents of which are hereby incorporated by reference in itsentirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. NIH 5R01DK060758-10 awarded by the National Institute of Health and the NationalInstitute of Diabetes and Digestive and Kidney Diseases. The Governmenthas certain rights in the invention.

All patents, patent applications and publications, and non-patentpublications cited herein are hereby incorporated by reference in theirentirety. The disclosures of these publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art as known to those skilled therein asof the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

Trefoil factor 2 (TFF2) is a small secreted protein that is expressed ingastrointestinal mucosa where it functions to protect and repair mucosa,but it is also expressed at low levels in splenic immune cells where itsrole has been unclear. The Tff2 gene is epigenetically silenced indigestive system cancers and thus has been postulated to protect againstcancer development through multiple mechanisms.

SUMMARY OF THE INVENTION

The present invention provides for the use of TFF2, delivered as arecombinant peptide or using a viral vector or modified peptide (forexample, a fusion protein for increased stability) to treat advancedcancer or dysplasia by specifically suppressing myeloid proliferationwith this approach. TFF2 can be a new and useful cancer therapy thatworks by targeting the tumor microenvironment, specifically the myeloidcells (e.g. MDSC, tumor associated macrophages, neutrophils) thatsupport cancer. In addition, it can be a form of replacement of a tumorsuppressor gene product that is normally downregulated in many cancers.

In one aspect, the invention provides for a method of treating orpreventing a cancer in a subject, the method comprising administering tothe subject a therapeutically effective amount of TFF2 protein, therebytreating or preventing the cancer. In one embodiment, the cancer iscolon cancer. In another embodiment, the cancer is rectal cancer. In oneembodiment, the cancer is gastric cancer. In another embodiment, thecancer is stomach cancer.

In one embodiment, the TFF2 protein is a human TFF2 protein. In oneembodiment, the TFF2 protein is a recombinant protein.

In one embodiment, the treating or preventing comprises inhibiting theproliferation of myeloid-derived suppressor cells (MDSCs).

In one aspect, the invention provides for a method of treating aninflammatory disease of the digestive system in a subject, the methodcomprising administering to a subject a trefoil family molecule. In oneembodiment, the inflammatory disease of the digestive system comprisesesophagitis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, colitis, irritable bowel syndrome, celiac disease, gastritis,or a combination thereof. In another embodiment, the inflammatorydisease of the digestive system is colitis.

In one aspect, the invention provides for a method of treating adigestive system cancer in a subject, the method comprisingadministering to a subject a trefoil family molecule. In one embodiment,the digestive system cancer is selected from the group consisting ofmouth cancer, pharynx cancer, esophageal cancer, and stomach cancer. Inanother embodiment, the digestive system cancer is selected from thegroup consisting of small intestine cancer, large intestine cancer,colon cancer, rectal cancer, and anal cancer. In another embodiment, thedigestive system cancer is selected from the group consisting of livercancer, pancreatic cancer, and gall bladder. In a further embodiment,the digestive system cancer is colon cancer.

In one aspect, the invention provides for a method of decreasing cellproliferation in a subject, the method comprising administering to asubject a trefoil family molecule, thus decreasing cell proliferation.In one embodiment, the cell is a myeloid-derived suppressor cell. Inanother embodiment, the myeloid-derived suppressor cell is a tumorassociated myeloid-derived suppressor cell. In another embodiment, themyeloid-derived suppressor cell is a myeloid-derived suppressor cellassociated with a tumor. In further embodiments, the myeloid-derivedsuppressor cell expresses a MDSC-specific surface marker. In oneembodiment, the myeloid derived suppressor cell does not express aMDSC-specific surface marker. In another embodiment, the surface markeris Grl1, CD11b, or a combination thereof. In another embodiment, thesurface marker is CD14, CD15, CD33, or a combination thereof. In anotherembodiment the surface marker is HLA-DR.

In one aspect, the invention provides a method of decreasing tumorgrowth in a subject, the method comprising administering to a subject atrefoil family molecule, thus decreasing tumor growth. In oneembodiment, the tumor is a tumor of the digestive system.

In one aspect, the invention provides a method of treating dysplasia ofthe digestive system in a subject, the method comprising administeringto the subject a trefoil family molecule.

In one aspect, the invention provides a kit for treating a trefoilfamily protein disorder, the kit comprising a trefoil family molecule,to administer to a subject and instructions of use.

In one aspect, the invention provides a method of determining thepresence of, or predisposition to, a trefoil family molecule disorder ina sample from a subject, the method comprising: (a) detecting thepresence, absence or reduction of a trefoil family molecule in thesample, wherein absence, or reduction of the molecule indicates thepresence of, or predisposition to, a trefoil family molecule disorder.In one embodiment, the method further comprises: (b) administering atrefoil family molecule to the subject where a trefoil family moleculewas not detected. In one embodiment, the method further comprisesincubating the sample with an agent that binds a trefoil familymolecule, or fragment thereof. In one embodiment, the agent is anantibody to a trefoil family molecule. In another embodiment, the sampleis digestive system cancer cells. In further embodiments, the digestivesystem cancer is selected from the group consisting of mouth cancer,pharynx cancer, esophageal cancer, and stomach cancer. In yet anotherembodiment, the digestive system cancer is selected from the groupconsisting of small intestine cancer, large intestine cancer, coloncancer, rectal cancer, and anal cancer. In another embodiment, thedigestive system cancer is selected from the group consisting of livercancer, pancreatic cancer, and gall bladder. In one embodiment, thedigestive system cancer is colon cancer.

In one embodiment, administering the trefoil family molecule isconducted simultaneously with the administering of a chemotherapy drug.In another embodiment, administering the trefoil family molecule isconducted sequentially in any order with the administering of achemotherapy drug. Non-limiting examples of conventional chemotherapydrugs include: aminoglutethimide, amsacrine, asparaginase, bcg,anastrozole, bleomycin, buserelin, bicalutamide, busulfan, capecitabine,carboplatin, camptothecin, chlorambucil, cisplatin, carmustine,cladribine, colchicine, cyclophosphamide, cytarabine, dacarbazine,cyproterone, clodronate, daunorubicin, diethylstilbestrol, docetaxel,dactinomycin, doxorubicin, dienestrol, etoposide, exemestane,filgrastim, fluorouracil, fludarabine, fludrocortisone, epirubicin,estradiol, gemcitabine, genistein, estramustine, fluoxymesterone,flutamide, goserelin, leuprolide, hydroxyurea, idarubicin, levamisole,imatinib, lomustine, ifosfamide, megestrol, melphalan, interferon,irinotecan, letrozole, leucovorin, ironotecan, mitoxantrone, nilutamide,medroxyprogesterone, mechlorethamine, mercaptopurine, mitotane,nocodazole, octreotide, methotrexate, mitomycin, paclitaxel,oxaliplatin, temozolomide, pentostatin, plicamycin, suramin, tamoxifen,porfimer, mesna, pamidronate, streptozocin, teniposide, procarbazine,titanocene dichloride, raltitrexed, rituximab, testosterone,thioguanine, vincristine, vindesine, thiotepa, topotecan, tretinoin,vinblastine, trastuzumab, and vinorelbine. In one embodiment, thechemotherapy drug is an alkylating agent (e.g. busulfan, cisplatin,carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine(DTIC), mechlorethamine (nitrogen mustard), melphalan, andtemozolomide), a nitrosourea, an anti-metabolite (e.g. 5-fluorouracil,capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine(ara-C), fludarabine, or pemetrexed), a topoisomerase inhibitor (e.g.topotecan, irinotecan, etoposide (VP-16), or teniposide), a mitoticinhibitor, an anthracycline (e.g. daunorubicin, doxorubicin(Adriamycin), epirubicin, idarubicin, or mitoxantrone), a corticosteroidhormone, a sex hormone, or a targeted anti-tumor compound (e.g. imatinib(Gleevec), gefitinib (Iressa), erlotinib (Tarceva), rituximab (Rituxan),or bevacizumab (Avastin)).

In one embodiment, administering the trefoil family molecule isconducted simultaneously with the administering of radiation therapy. Inanother embodiment, administering the trefoil family molecule isconducted sequentially in any order with the administering of radiationtherapy. Non-limiting examples of conventional radiation therapyinclude: external beam radiation therapy, sealed source radiationtherapy, unsealed source radiation therapy, particle therapy, andradioisotope therapy.

In one embodiment, administering the trefoil family molecule isconducted simultaneously with the administering of cancer immunotherapy.In another embodiment, administering the trefoil family molecule isconducted sequentially in any order with the administering of cancerimmunotherapy. Non-limiting examples of cancer immunotherapy include:cancer vaccines, therapeutic antibodies, such as monoclonal antibodytherapy (e.g., Bevacizumab, Cetuximab, and Panitumumab), cell basedimmunotherapy, and adoptive cell based immunotherapy.

In one embodiment, administering the trefoil family molecule isconducted simultaneously with the administering of an anti-inflammatorydrug. In another embodiment, the trefoil family molecule is conductedsequentially in any order with the administering of an anti-inflammatorydrug. Non-limiting examples of anti-inflammatory drugs include:anti-inflammatory steroids (corticosteroids) (e.g. prednisone),aminosalicylates (e.g., mesalazine), non-steroidal anti-inflammatorydrugs (NSAIDs) (e.g. aspirin, ibuprofen, naproxen) and immune selectiveanti-inflammatory derivatives (ImSAIDs). An anti-inflammatory drug alsoincludes antibodies or molecules that target cytokines and chemokinesincluding, but not limited to, anti-TNFα antibodies (e.g. infliximab(Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab(Simponi), etanercept (Enbrel)), anti-IL12 antibodies, anti-IL2antibodies (basiliximab (Simulect), daclizumab (Zenapax), azathioprine(Imuran®, Azasan®), 6-mercaptopurine (6-MP, Purinethol®), cyclosporine A(Sandimmune®, Neoral®), tacrolimus (Prograf®), and anti-GM-CSFantibodies.

In one aspect, the invention provides a diagnostic kit for determiningthe presence of, or predisposition to, a trefoil family moleculedisorder, the kit comprising an agent that binds to a trefoil familymolecule, and instructions for use. In one embodiment, the agent is anantibody to a trefoil family molecule.

In one embodiment, the trefoil family molecule is TFF1. In anotherembodiment, the trefoil family molecule is TFF2. In a furtherembodiment, the trefoil family molecule is TFF3.

In one embodiment, the subject is a human. In another embodiment, thesubject is a cat. In a further embodiment, the subject is a dog.

In one embodiment, the trefoil family molecule is a nucleic acid. Inanother embodiment, the nucleic acid is delivered as a viral vector. Inone embodiment, the nucleic acid comprises the nucleotide sequence ofSEQ ID NO: 2. In another embodiment, the nucleic acid comprises thenucleotide sequence of SEQ ID NO: 4. In a further embodiment, thenucleic acid comprises the nucleotide sequence of SEQ ID NO: 6.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-D. TFF2 is expressed in splenic T-cells and upregulated uponT-cells activation. FIG. 1A. Detection of TFF2 protein in the mousestomach and spleen by western blot analysis. Detection of TFF2 in spleenby immunoprecipitation. Total splenic extracts from five wild type micewere combined and used for this experiment. A half of extracts wasprecipitated by TFF2 antibodies while another half was precipitated byIgG isolated from pre-immune serum. Protein A—bounded with agarose beadswas added and incubated for 24 h at 4° C. Precipitates were resolved ingel-electrophoresis, and revealed by WB analysis using anti-mouse TFF2antibodies. Stomach extracts from wild-type mouse was used as positivecontrol. The position of TFF2 is indicated by arrow, positions ofmolecular weight markers (Mr, kDa) are shown on the right. FIG. 1B.Detection of TFF2 protein in the mouse stomach and spleen by westernblot analysis. Detection of TFF2 in mouse tissue extracts (stomach,spleen, thymus) by Western blot by using SuperSignal® West Femto TrialKit. The position of TFF2 is indicated by arrow, positions of molecularweight markers (Mr, kDa) are shown on the left. FIG. 1C. Detection ofTFF2 mRNA in T-cells by semi-quantitative RT-PCR. Total mRNA wasisolated from splenic T and B cells (lanes T and B, top panel), reversetranscribed and amplified by PCR by using primers, which are specificfor mTFF2 (top panel); T-cells marker Thy 1.2 (second panel); B-cellsmarker CD19 (third panel); actin (loading control, bottom panel). Theresulted products were resolved by agarose gel electrophoresis as shown.The positions of 100 bp DNA ladders (M) are shown on the right. FIG. 1D.Upregulation of TFF2 mRNA expression by mitogenic stimuli in mousesplenocytes. Mouse splenocytes were activated by concavalin A (ConA, 1μg/ml), bacterial lipopolysaccharides (LPS, 100 ng/ml) or leftunstimulated (unst) for 24 h. The relative abundance of TFF2 mRNA wasmeasured in the splenocytes and stomach (positive control) by real-timePCR.

FIG. 1E-G. TFF2 is upregulated in spleen upon DSS treatment. FIG. 1EWestern blot of splenic tissues obtained from TG mice after DSSadministration. Time-course change of TFF2 peptide in spleen upon DSS inCD2-TFF2 transgenic mice FIG. 1F RT-PCR analysis of WT mice spleentissues during administration of 3% DSS water showed increased levels ofTFF2 mRNA expression. TFF2 is increased in spleen of WT mice upon DSSadministration. FIG. 1G Western blot of splenic tissues obtained fromwild type mice after DSS administration. Time-course change of TFF2peptide in spleen upon DSS in wild type mice.

FIGS. 2A-B. Generation and characterization of transgenic CD2-TFF2 mice.FIG. 2A. Scheme of cloning TFF2 into the hCD2-TFF2 cassette. Thefull-length gene of murine TFF2 was cloned into a unique EcoRI site ofthe hCD2 minigene cassette, which contains the promoter, polyadenylationsite, and locus control region elements from the human CD2 gene. FIG.2B. RT-PCR analysis of TFF2 mRNA expression under control of hCD2promoter in spleen and thymus of CD2-TFF2 transgenic mice. Upper panel,the spleen and thymus cells of the offspring of founders possessedCD2-TFF2 mRNA, 1,2—spleen and thymus from wild type, 3 and 4, 5 and6—spleen and thymus accordingly from transgenic mice, 7,8—water. Middlepanel, controls consisted of samples without reverse transcriptase werenegative for presence of CD2-TFF2 mRNA. Bottom panel, the expressionlevel of TFF2 mRNA was normalized for GAPDH mRNA.

FIG. 2C is a western blot showing expression of TFF2 peptide in spleenand thymus of unchallenged CD2-TFF2 transgenic mice. Cell extracts fromwhole spleen and thymus were immunoblotted with anti-TFF2 antibodies,stomach extract from wild type mice was used as a positive control.

FIGS. 3A-E. TFF2-deficiency results in more severe inflammation upon DSSchallenge. FIG. 3A. Kaplan-Meier survival curves of TFF2−/−, TG and WTmice administered 3% DSS. Representative data of one of two independentexperiments (n=12 per TFF2−/−, n=8 per WT, n=7 per TG mice). FIG. 3B.Colitis was induced in mice by administration of 3% DSS in drinkingwater for 5 days, mice were sacrificed and analyzed on day 19.Quantification of colon size of wild type, transgenic and TFF2−/− mice.FIG. 3C. Representative spleen appearance of transgenic, wild type andTFF2−/− mice upon 2.5% DSS treatment. FIG. 3D. Representative colonappearance of wild type, transgenic and TFF2−/− mice upon 2.5% DSStreatment. FIG. 3E. Quantification of spleen mass of transgenic, wildtype and TFF2−/− mice upon 2.5% DSS treatment.

FIGS. 3F-G. TFF2-deficiency results in more severe inflammation upon DSSchallenge. FIG. 3F. Microscopic evaluation of DSS-induced colon damage.WT, TFF2−/− and TG mice were administrated 2.5% DSS for 5 days followedby tap water for the next two weeks. Colons were obtained on day 19 fromeach group. Results are representative of two independent experiments(4-6 animals per group). Colons were stained with haematoxylin andeosin. Panels: upper—TFF2−/−, middle—WT, bottom—TG, no DSS (left),atrophy and inflammation upon DSS treatment (right). FIG. 3G.Inflammatory score of colon tissue after DSS treatment (day 19).

FIGS. 4A-D. Immune response in transgenic, wild type and TFF2−/− mice.Colitis was induced by addition of 2.5% DSS to drinking water for 5days, mice were sacrificed on day 19; colonic tissues were used foranalysis of cytokines IL-1beta, TNF alpha, IFN-gamma and IL-6 by ELISA.Cytokines levels of IL-1beta (FIG. 4A), IL-6 (FIG. 4B), IFN-gamma (FIG.4C) and TNF alpha (FIG. 4D) normalized to protein content of colonictissues. Data represent results of 2-3 independent experiments with 5-8mice per group.

FIG. 4E is a plot showing that TFF2-deficient mice display higher levelof myeloperoxidase (MPO) activity at acute phase of colitis (day 6).

FIGS. 5A-D. Influence of TFF2 expressed in immune compartment onsystemic and colonic inflammation. Effect of TFF2 expressed in thelymphohematopoietic compartment on systemic and colonic inflammation.Lethally-irradiated wild type mice were transplanted with bone marrowfrom WT, TFF2−/− and TG animals. Eight weeks later chimeric animalsreceived 3% DSS water for 5 days followed by tap water for 2 weeks. Micewere analyzed on day 19. Differences in clinical disease parametersbetween chimaeras transplanted with bone marrow from donor TFF2−/−, WTand TG mice: body weight loss (a statistically significant difference(*, P<0.05) in body weight loss is marked by an asterisk.) (FIG. 5A),shrinking colon in mice transplanted with bone marrow from donor TFF2−/−animals (FIG. 5B), lower spleen mass in chimaeras transplanted with bonemarrow from donor TG mice at day 6 and 19 (FIG. 5C) and splenomegaly atday 19 (FIG. 5D) in chimaeras wild type mice receiving bone marrow fromdonor TFF2−/− counterparts.

FIGS. 5E-F. Influence of TFF2 expressed in immune compartment onsystemic and colonic inflammation. Plots show the level of IL-1β (FIG.5E) and IL-6 (FIG. 5F) in colon tissues at day 19 in ELISA.

FIGS. 5G-H show histology (G) and inflammatory score (H) of colon ofwild type transplanted with bone marrow from donor wild type, transgenicand SPKO mice on days 6 and 19 (FIG. 5G). Histology, upper panel, day 6:donor WT and TG mice, atrophy; donor TFF2−/− mice, atrophy (a) andinfiltration of inflammatory cells under mucosa (b). Bottom panel, day19: donor WT and TG mice, atrophy; donor TFF2−/− mice, atrophy (a) andinfiltration of inflammatory cells under mucosa (b). Magnification ×200.Histological score on day 19 (FIG. 5H).

FIGS. 6A-G show the effect of TFF2 expressed in immune cells on colonand systemic inflammation upon DSS challenge in TFF2−/− mice substitutedwith bone marrow from wild type, TFF2−/− and TG mice. All groups weregiven 2.5% DSS water for 5 days followed by tap water. Mice weresacrificed on day 19. Clinical disease parameters between TFF2−/− micetransplanted with bone marrow from TFF2−/−, WT and TG animals: bodyweight change (FIG. 6A), chimaeras mice received bone marrow fromTFF2−/− animals have higher diarrhea (FIG. 6B) and bleeding score (FIG.6C) compare with mice receiving bone marrow from TG animals, colonlength (FIG. 6D), splenomegaly in TFF2−/− mice that received bone marrowfrom TFF2−/− mice and normal mass of spleen in TFF2−/− mice receivedbone marrow from TG mice (FIG. 6E). ELISA for IL-1b in colon tissues(FIG. 6F). Myeloperoxidase activity at day 6 (FIG. 6G).

FIG. 6H are plots that show the survival rate of wild type (upper panel)and TFF2−/− (lower panel) chimaeras with bone marrow transplanted fromWT, TG and TFF2−/− mice (FIG. 6H).

FIGS. 7A-B. Accumulation of CD11b+Gr1+ cells in spleens from DSS-treatedTFF2−/− and WT mice. FIGS. 7A-B. show immunohistochemical staining ofspleens from TFF2−/−, WT and TG mice on day 19 after DSS treatment.Spleens are stained with anti-Ki67 (nuclear proliferating antigen,brown) and anti-Gr1 (red) antibodies. Note an expansion of red pulp anda partially overlapped staining for Gr1 and Ki67 markers in spleen ofTFF2−/− mouse Magnification ×40 (FIG. 7A) and ×200 (FIG. 7B).

FIG. 7C. TFF2 suppresses expansion of CD11b+Gr1+ cells in spleen andbone marrow upon DSS treatment. Flow cytometry analysis of CD11b1+staining versus Gr1+ staining gated on live cells from the spleen ofspleen of mice with DSS colitis on Day 19 (splenic cells—upper row, bonemarrow cells—lower row). Numbers indicate percentage of CD11b+Gr1+.

FIGS. 7D-E. Graphical representation of FACS analysis data (D).Percentages of CD11b+Gr1+ cells from spleen (left) and bone marrow(right). Data represent the mean and standard deviation of 3-6 animalsper group. Asterisks P<0.05 by analysis of variance and the Student'stest. Mice were given 3% DSS water for 5 days, and then they switched totap water and sacrificed at indicated time points. FIG. 7E. BrdU uptakeby Gr1+CD11b+ cells from TFF2−/−, wild type and transgenic mice.

FIG. 7F is a bar graph of a CFU assay of splenocytes from TG, WT andTFF2−/− mice after DSS treatment. Bar graphs enumerate colonies incultures.

FIGS. 8A-C. Recombinant mouse TFF2 inhibits proliferation of sortedGr1+CD11b+ cells in vitro. Spleens were obtained from TFF2−/− on day 19after starting DSS water. Splenic cells from TFF2−/− mice were labeledfor Gr1 and CD11b antigen and sorted on sorter FACSAria. FIG. 8A TFF2inhibits BrdU uptake by Gr1+CD11b+ cells. Isolated Gr1+CD11b+ cells(2×10⁵/well) were cultured 7 days in presence of 10 or 5 ng/ml ofGM-CSF. FIG. 8B Effect of TFF2 protein on Gr1+CD11b+ cell survival anddeath. Sorted Gr1+CD11b+ cells (more then 95% of pure cells) were grownwith 5 ng/ml of GM-CSF with addition of TFF2 in indicated concentrationsfor 8 days. FIG. 8C Proliferation was measured in triplicate by BrdUuptake after 24 h pulse and expressed in arbitrary units (AU at 450 nm).

FIG. 9A shows phenotypical characterization of Gr1+CD11b+ cells.Morphology of sorted Gr1+CD11b+ cells. Splenocytes cells from TFF2−/−mice with DSS treatment were labeled for Gr1 and CD11b antigen andsorted on sorter FACSAria. Purity of sorted cells was more than 95%.

FIG. 9B shows phenotypical characterization of Gr1+CD11b+ cells.Phenotype of Gr1+CD11b+ cells. Cells were stained for indicated antigenfor flow cytometry analysis. Cells were gated on viable Gr1+CD11b+population and expression of various markers was analyzed. Isotypecontrol is shown as shaded and antigen staining as unshaded histogramaccordingly. Representative data on antigen expression of sortedGr1+CD11b+ cells.

FIG. 9C is a plot showing sorted Gr1+CD11b+ cells loose expression ofGr1 antigen while increase CD11c marker during culture with GM-CSF (10ng/ml).

FIG. 9D is a plot showing sorted splenic Gr1+CD11b+ cells from TFF2−/−mice, followed by staining for F4/80 or CD11c. Only a small proportionof the cells stain for F4/80 while the majority of the cells arepositive for CD11c.

FIGS. 10A-B. Recombinant mouse TFF2 inhibits proliferation of sortedGr1+CD11b+ cells in vitro. Spleens were obtained from TFF2−/− on day 19after starting DSS water. Splenic cells from TFF2−/− mice were labeledfor Gr1 and CD11b antigen and sorted on sorter FACSAria. FIG. 10A. TFF2inhibits BrdU uptake by Gr1+CD11b+ cells. Isolated Gr1+CD11b+ cells(2×10⁵/well) were cultured 7 days in presence of 10 or 5 ng/ml ofGM-CSF. Proliferation was measured in triplicate by BrdU uptake after 24h pulse and expressed in arbitrary units (AU at 450 nm). FIG. 10B.Effect of TFF2 protein on Gr1+CD11b+ cells survival and death. SortedGr1+CD11b+ cells (more than 95% of pure cells) were grown with 5 ng/mlof GM-CSF with addition of TFF2 in indicated concentrations for 7 days.TFF2 directly suppresses growth by IMC in response to GM-CSF, in vitroIMCs were sorted from spleen of TFF2−/− mice treated with DSS andcultured in presence of GM-CSF for 7 days.

FIGS. 11A-B. TFF2−/− mice are more susceptible while TG mice are moreresistant to azoxymethane-DSS-induced colonic tumorigenesis. FIG. 11ARepresentative colons of TFF2−/−, WT and TG (upper, middle and lowerpanels accordingly) mice after AOM/DSS treatment at 5 months. FIG. 11BTumor burden of TFF2−/−, WT and TG mice. Each group of mice included 4-7animals, 3 independent experiments were done.

FIG. 11C is a photomicrograph of H&E stained colonic tumors in TFF2−/−,wild type and CD2-TFF2 mice.

FIG. 11D is a plot showing that AOM+DSS treatment resulted into higheraccumulation of Gr1+CD11b+ cells in bone marrow, spleen and blood inTFF2−/− mice compare with wild type and transgenic animals. Spleen, bonemarrow and blood were harvested from mice and analyzed for Gr1+CD11b+cells. Representative dot plots of flow cytometry data of CD11b1+staining versus Gr1+ staining gated on live cells. Proportions ofGr1+CD11b+ cells in spleen, blood and bone marrow. Data are pooled from5-6 mice of each group of mice and represent data from 3 independentexperiments.

FIG. 11E is a plot showing CD11b−Ly6C+, CD11b+Ly6C− and CD11b+Ly6G−cells in tumor tissue. Tumor from TFF2−/− mice was digested withcollagenase IV, cells were stained with antibody CD11b, Ly6C, Ly6G andF4/80 and analyzed by flow cytometry.

FIG. 11F-I are plots showing gene expression was normalized on GAPDHlevels and the expression of each gene relative to untreated colontissues of wild type mice is depicted. Il-1β (FIG. 11F) and IL-6 (FIG.11G) level in tumor tissues obtained from TFF2-deficient, TG and wildtype mice. Il-1β (FIG. 11H) and IL-6 (FIG. 11I) level and in colonuninvolved in tumor obtained from TFF2-deficient, TG and wild type mice.The data correspond to a representative experiment out of threeexperiments.

FIG. 12. TFF2−/− deficient mice expand myeloid progenitor cells inspleen (and bone marrow). Identification of GMP cells in spleen ofTFF2−/− deficient, WT and TG mice after AOM/DSS treatment(representative data from 4-5 mice of each group).

FIGS. 13A-D. Relative mRNA expression of IL-1beta (FIG. 13A), TNF-alpha(FIG. 13B), IFN-gamma (FIG. 13C) and IL-6 (FIG. 13D) in colon tissues.Colitis was induced by addition of 2.5% DSS to drinking water for 5days, mice were sacrificed on day 19; colonic tissues were used foranalysis of mRNA cytokines IL-1beta, TNF alpha, IFN-gamma and IL-6. Datarepresent results of 2-3 independent experiments with 5-8 mice pergroup.

FIGS. 14A-B are plots showing FACS analysis. FIG. 14A are plots showingFACS analysis of splenic CD11b+GR1+ cells in WT and CD2-TFF2 transgenic(TG) mice at 14 days and 19 days after starting DSS treatment. WT miceshow a significant increase in IMCs while TG mice do not. FIG. 14B.shows FACS analysis for FOXP3+ cells in the spleens of WT and TG miceafter DSS treatment.

FIG. 15 shows photomicrographs of hematoxylin and eosin-stained spleen(frozen tissue) from TFF2−/−, WT and TG mice (Magnification 60×). Micewere treated with DSS water and spleen was stained for hematoxylin andeosin. Spleen from TG mice displays lowest proportion cells with ringshaped nuclei compare with WT and TFF2−/− mice.

FIG. 16A. Gr1+ and CD11b+ cells from spleen of transgenic, wild type andTFF2−/− deficient mice proliferate during recovery phase of DSS-inducedcolitis. Proliferative capacity was assessed by BrdU uptake on Gr1+ andCd11b+ cells from spleen taken from mice treated with 3% DSS on day 19.Cells were analyzed 72 h after first injection of BrdU.

FIG. 16B. Gr1+ and CD11b+ cells from spleen of transgenic, wild type andTFF2−/− deficient mice proliferate during recovery phase of DSS-inducedcolitis. Gating strategy for analysis of myeloid progenitor cells (GMP).Dead cells were excluded from analysis by staining cells with (DAPI).DAPI-negative population was gated inside of lineage-negative (Lin−)cells along with IL-7Rα-negative cells, and then cells were analyzed forprogenitors cells. GMP cells were identified as Lin−IL-7Rα− ckit+ cells,CD34+ CD16/32+ population within ckit+Sca− cells.

FIG. 17. are bar graphs showing TFF2 is up-regulated in spleen upon DSStreatment.

FIG. 18. Gating strategy for sorting of CD4+ memory T+cells. Cell weregated on CD4+ live cells, then CD44^(low)CD62L^(high) naïve andCD44^(high)CD62L^(low) memory cells were sorted and total mRNA fractionwas extracted.

FIG. 19. TFF2 mRNA is up-regulated in splenic memory CD4+ T-cells ofwild type mice upon DSS treatment. Wild type mice were challenged with6% DSS for 48 h. Messenger RNA from total, naïve and memory CD4 T cellswas purified and subjected Taqman RT-PCR analysis with TFF2 and GAPDHspecific primers and probes. Relative TFF2/GAPDH abundance is plotted onthe graph.

FIG. 20. Vagotomy results in TFF2 mRNA down-regulation in spleen of micetreated with DSS. Mice with vagotomy (VTPP) group and control micewithout vagotomy (Sham) were given 6% for 48 h DSS water.

FIG. 21. is a plot showing that vagal nerve stimulation (VNS) does notchange level TFF2 mRNA in spleen of untreated mice.

FIG. 22. Change of body weight over 19 days of TFF2−/−, WT and CD2-TFF2mice. Mice were given 3% DSS in drinking water during 5 days, then micewere given tap water for 14 days. On day 19 mice were sacrificed.

FIG. 23. DSS treatment results in splenomegaly in TFF2−/− mice.Quantification of spleen mass of transgenic, wild type and TFF2−/− miceupon DSS treatment.

FIG. 24. TG mice are more resistant to AOM/DSS. Representative colons ofTFF2/−, WT and TG (upper, middle and lower panels accordingly) miceafter AOM/DSS treatment (left) and tumor burden of TFF2/−, WT and TGmice (right).

FIG. 25. are bar graphs depicting that TG mice show lowest proportion ofCD11b+Gr1+ cells in spleen and bone marrow.

FIGS. 26A-B. Representative flow cytometry dot plots showing thepercentage of CD11b+Gr1+ cells in tumor tissue of TFF2−/− mice 5 monthsafter AOM/DSS regimen. FIG. 26A. Tumor from TFF2−/− mice was digestedwith collagenase IV and DNAase I. CD11b+Gr1+ were gated on live CD45+cells. FIG. 26B. Preponderance of CD11b+Ly6G+ population in colon tumortissue of TFF2−/− mice. Single cells from tumor were obtained as above.Cells were gated on Ly6C and Ly6G positive subsets on CD11b+ populationamong CD45+ live cells.

FIG. 27. Survival rate of TFF2−/−, CD2-TFF2, and wild type mice upon 3%DSS treatment. Mortality was assessed as the primary endpoint.Differences in survival curves between two groups were analyzed usingthe Log-rank (Mantel Cox) test. Representative data of one of twoindependent experiments (n=12 per TFF2−/−, n=8 per WT, n=7 per TG mice).

FIG. 28. Change of spleen mass of TFF2−/−, WT and CD2-TFF2 mice. Micewere given 3% DSS in drinking water during 5 days, then mice were giventap water for 14 days. On day 19 mice were sacrificed.

FIG. 29. Mice with bone marrow transplanted from TFF2−/mice develop moretumors compare with wild type and CD2-TFF2 transgenic mice. Wild typemice were lethally irradiated and bone marrow from wild type, CD2-TFF2and TFF2−/mice was transplanted. Animals were submitted AOM/DSStreatment and analyzed after 5 months.

FIG. 30 is a bar graph showing increased CFU in spleen of TFF2−/− butnot in CD2-TFF2 transgenic mice treated with DSS, day 19.

FIG. 31 is a plot showing increased GMP progenitor number in spleen ofTFF2−/− but not in CD2-TFF2 transgenic mice treated with DSS, day 19.

FIG. 32 is a plot showing increased GMP progenitor number in spleen ofTFF2−/− but not in CD2-TFF2 transgenic mice treated AOM/DSS

FIG. 33 is a bar graph showing CD11b+Gr1+ cells suppress BrdU uptake bypolyclonally activated CD4+ T-cells.

FIG. 34 is a bar graph showing that CD11b+Gr1+ cells suppress INF-γproduction by polyclonally activated CD4+ T-cells.

FIG. 35 is a bar graph showing that CD80 Ab abrogate the suppressiveeffect of CD11b+Gr1+ cells.

FIG. 36. are plots showing phenotype characterization and increase ofCD80 marker expression on MDSCs after AOM/DSS treatment in spleen ofTFF2−/− mice.

FIG. 37. TFF2 directly suppresses BrdU uptake by IMC in response toGM-CSF. in vitro IMCs were sorted from spleen of TFF2−/− mice treatedwith DSS and cultured in presence of GM-CSF for 7 days.

FIG. 38 is a bar graph showing recombinant TFF2 suppresses CD11b+Gr1+cells from spleen of tumor-bearing mice.

FIG. 39 shows plots that depict splenic CD11b+Gr1+ cells from TFF2−/−mice show higher capacity for proliferation compare with IMCs from wildtype and CD2-TFF2 mice. BrdU was injected 3 h before sacrifice.

FIG. 40 is a bar graph (top) and plot (bottom) showing TFF2-deficiencyresults in a higher number of splenic CFU and GMP in the DSS model.

FIG. 41 is a bar graph (top) and plot (bottom) showing TFF2-deficiencyresults in a higher number of splenic CFU and GMP in the AOM/DSS model.

FIG. 42 is a schematic of a microarray analysis of DSS-induced splenicCD11b+Gr1+ cells workflow.

FIG. 43 shows images of western blots of mouse recombinant TFF2 that isexpressed by Ad-TFF2 system and secreted in medium.

FIG. 44 shows a schematic of seven transcription factors upregulated(>3-fold) by TFF2 in CD11bGr1 cells.

FIG. 45. qPCR Analysis. TFF-2 down-regulates cyclin D1, cyclin E1 andCD11c antigen expression in CD11b+Gr1+ cells.

FIG. 46. FACS Analysis. TFF-2 down-regulates cyclin D1, cyclin E1 andCD11c antigen expression in CD11b+Gr1+ cells.

FIG. 47. Diagram of the adenoviral construct expressing tagged TFF2 andevidence for expression of the recombinant product. The full-lengthmTFF2 cDNA fragment was generated through PCR amplification using thepCMV3-mTFF2 construct, then was subcloned into pAdlox vector to generatethe pAdlox-mTFF2 construct and packaged in HEK293 cells (top). TheAd-mTFF2 constructs were verified by PCR and DNA sequencing (bottom).

FIG. 48. Recombinant mTFF2 expression in cultured cells. The efficiencyof Ad-GFP (80%)/Ad-mTFF2 (60%) infection were identified byimmunofluorescence in AGS cells.

FIG. 49. SDS gel showing purified recombinant mTFF2 protein.

FIG. 50. Expression of murine TFF2 in 293 cells and gastric cancer celllines. Western blot showing recombinant mTFF2 expression in 293 cells,after Adlox-mTFF2 construct transfection or Ad-GFP infection (top).Western blot showing recombinant mTFF2 expression in AGS/MKN28 cells,after Ad-mTFF2 or Ad-GFP infection (bottom).

FIG. 51. Recombinant mTFF2 expression in vivo. Recombinant mTFF2expression in stomach, liver and spleen of WT or TFF2−/− mice with andwithout Ad-mTFF2 injection. Ad-GFP was used as a control to show thetarget tissue of Ad-virus. Immunohistochemical and GFP fluorescenceevidence for TFF2 expression in the stomach, liver and spleen of WT andTFF2 deficient mice.

FIG. 52. Recombinant mTFF2 expression in vivo. After one dose ofAd-mTFF2 injection (5×10⁸ per mouse), a high level of mTFF2 was found inthe serum from NOD-SCID, WT, and TFF2−/− mice, respectively.

FIG. 53. Recombinant mTFF2 expression in vivo. In NOD-SCID mice, highlevels of serum mTFF2 were maintained for at least 7 weeks. In WT mice,levels were maintained for at least 3 weeks.

FIG. 54. Recombinant mTFF2 expression in vivo. In TFF2−/− mice, highlevels of serum mTFF2 were maintained for 2 weeks.

FIG. 55 shows an image of mice spleens and a bar graph depicting thequantitation of spleen size after Ad-TFF2 treatment of TFF2−/− miceafter AOM/DSS treatment.

FIG. 56. Effects of adenoviral TFF2 on splenic, BM and circulating IMCs.The time point of Ad-TFF2 injection. The first dose was given at 2 weeksafter AOM treatment, then, Ad-mTFF2 treatment was repeated every 3weeks. Delivery of adenoviral TFF2 to AOM/DSS treated mice suppressedMDSCs. FACS data showing reductions in CD11b+Gr1+ myeloid cells in theblood, spleen and bone marrow after adenoviral delivery of TFF2. Ad-TFF2treatment significantly alleviated the elevated proportion of Gr1+CD11b+cells, which were induced by AOM/DSS treatment, not only in the spleenand peripheral blood from wild-type mice, but also in those from TFF2−/−mice.

FIG. 57. Suppression of colon tumors by Ad-TFF2. Gross photographs andquantitation of tumor number after Ad-TFF2 treatment of TFF2−/− miceafter AOM/DSS treatment (top). 13 weeks after AOM/DSS induction, thenumber of tumors in the colon of Ad-TFF2 treated mice is much less thanthat in control mice, especially in TFF2−/− mice (bottom).

FIG. 58. mRNA level of IL-6 in tumor tissues obtained fromTFF2-deficient, TG and wild type mice. Gene expression was normalized onGAPDH levels and the expression of each gene relative to untreated colontissues of wild type mice is depicted.

FIGS. 59A-D. mRNA level of IL-1β (FIG. 59A) and IL-6 (FIG. 59B) in colonwithout visible tumor obtained from TFF2-deficient, TG and wild typemice. Gene expression was normalized on GAPDH levels and the expressionof each gene relative to untreated colon tissues of wild type mice isdepicted. FIG. 59C. Higher level of IL-1 β in tumor tissue compare withcolon without visible in tumor in TFF2−/− mice. FIG. 59D. Flow cytometryanalysis of Ly6C+ and Ly6G+ cells after gating on CD11b+ cells in spleenof tumor-bearing TFF2−/− mice.

FIG. 60. CD11b+Gr1+ cells expand temporally in spleen and bone marrowupon DSS treatment. Bars represent percentages of CD11b+Gr1+ cells amonganalyzed live cells. The means and standard deviations were calculatedbased on data for 3-6 animals per group. Asterisks indicate astatistically significant difference (*P<0.05, ***P<0.001) in CD11b+Gr1+cell number between tested groups.

FIGS. 61A-E are plots and graphs showing FACS analysis of CD11b+Gr1+splenic cells for other MDSC markers, including F4/80, CD11c, CD115,CD31, MHCII, CD86, CD40 and CD80 (FIGS. 61A, 61C). FIG. 61B showsanalysis by FACS of Ly6C versus Ly6G cells. Both are suppressed in TGmice. FIG. 61D shows the effect of MDSCs on CD4 proliferation andIFNgamma secretion in response to stimulation.

FIG. 62. Splenic CD4+ T cells were sorted and then placed in culture andstimulated with 50 or 500 nM of isoproterenol and then assayed for TFF2mRNA expression by RT-PCR.

FIGS. 63A-B. (A) FACS plots of CD4+ memory T cells, demonstrating strongEGFP expression in these cells. (B) Immunofluorescence ofCD4+CD44hiCD62Llo memory T cells with antibodies to CD44 (green) andTFF2 (red) showing co-localization.

FIG. 64. TFF2 mRNA expression in immune cells in the spleen.

FIG. 65. CD8 T-cells play role in TFF2-mediated protection of colon fromtumor development. CD2-Tff2 mice were injected with AOM following DSStreatment. Four weeks later CD8 antibody (300 ug/mouse) was injectedintraperitoneally twice a week and mice were sacrificed 12 weeks later.(Right) bars present number of tumors/mouse in mice received CD8 Ab vsmice received isotypic control.

FIG. 66. Increased activated CD8+ T cells in the spleens of CD2-TFF2transgenic mice. ELISPOT for IFN-g and Granzyme B from sorted CD8+ Tcells from CD2-TFF2 and TFF2−/− mice after AOM/DSS.

FIGS. 67A-B. MDSCs contribute to carcinogenesis in AOM/DSS-induced coloncancer model. Adoptive transfer of MDSC results into tumor progressionin CD2-Tff2 mice. CD2-Tff2 mice were injected with AOM following DSSwater for 7 days. After 4 weeks 5 mice were subjected intravenous tailinjection once a week with 2-3×10⁶ MDSC sorted from spleen and bonemarrow from tumor-bearing Tff2-null mice and 5 mice from control groupreceived PBS injection. (A) representative pictures of colon fromcontrol group and group received MDSC. (B) bars present number oftumor/mouse in mice received MDSC vs. mice received PBS.

FIGS. 68A-B. Splenic IMC from Tff2-null mice show higher contribution intumorigenesis vs. splenic IMC from CD2-Tff2 mice. WT CD45.1 mice weretreated with AOM/DSS, 13 weeks after, recipient mice were infused with3×106 CD11b+Gr1+ cells sorted from either Tff2-null or CD2-tff2 micespleen. 5 weeks after adoptive transfer, recipient mice were killed,colon tumor number were counted, MDSCs and CD8+ T cells were analyzed byFACS. Wildtype mice that received IMC from spleen of Tff2-nulls micedevelop more tumors in colon and bigger spleens (A) and lower CD8T-cells in colon tissues (B).

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The singular forms “a”, “an” and “the” include plural reference unlessthe context clearly dictates otherwise. The use of the word “a” or “an”when used in conjunction with the term “comprising” in the claims and/orthe specification may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

The invention is directed to methods of treating diseases of thedigestive system in a subject comprising administering a trefoil familymolecule. For example, the invention is directed to methods for treatingan inflammatory disease of the digestive system in a subject. Theinvention is also directed to methods for treating a digestive systemcancer. The invention is also directed to methods of decreasing tumorgrowth. The invention is also directed to treating dysplasia of thedigestive system. The invention further encompasses methods ofdecreasing cell proliferation in a subject comprising administering atrefoil family molecule. For example, the invention is directed tomethods of decreasing the proliferation of myeloid-derived suppressorcells.

As would be apparent to one of ordinary skill in the art, any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

Diseases of the Digestive System

The present invention provides methods for treating diseases of thedigestive system. In one embodiment the digestive system comprises thegastrointestinal tract including structures from the mouth to the anus,and the accessory organs. For example, this includes, but is not limitedto, the mouth, the pharynx, the esophagus, the stomach, the smallintestine, including the duodenum, jejunum, and ileum, the largeintestine including the cecum, colon, and rectum, and the anus. Infurther embodiments, the accessory organs of the digestive systeminclude, but are not limited to, the liver, the pancreas, and the gallbladder.

The present invention provides methods for treating an inflammatorydisease of the digestive system in a subject comprising administering atrefoil family molecule. In one embodiment, the inflammatory disease ofthe digestive system includes, but is not limited to, esophagitis,inflammatory bowel disease, Crohn's disease, ulcerative colitis,colitis, irritable bowel syndrome, celiac disease, and gastritis.

In some embodiments, the subject is already suspected to have aninflammatory disease of the digestive system. In other embodiments, thesubject is being treated for an inflammatory disease of the digestivesystem, before being treated according to the methods of the invention.In other embodiments, the subject is not being treated for aninflammatory disease of the digestive system, before being treatedaccording to the methods of the invention.

The present invention provides methods for treating a digestive systemcancer in a subject comprising administering a trefoil family molecule.In one embodiment, the digestive system cancer includes, but is notlimited to, mouth cancer, pharynx cancer, esophageal cancer, stomachcancer, small intestine cancer, large intestine cancer, colon cancer,rectal cancer, anal cancer, liver cancer, pancreatic cancer, and gallbladder cancer.

In some embodiments, the subject is already suspected to have adigestive system cancer. In other embodiments, the subject is beingtreated for a digestive system cancer, before being treated according tothe methods of the invention. In other embodiments, the subject is notbeing treated for a digestive system cancer, before being treatedaccording to the methods of the invention.

The present invention also provides methods for decreasing tumor growthin a subject comprising administering a trefoil family molecule. In oneembodiment, the tumor is a tumor of the digestive system. Tumor growthcan be measured in a variety of ways, known to one of skill in the art.For example, tumor growth can be measured by measuring the tumor volumeover time. Tumor volume can be measured in a variety of ways, known toone of skill in the art including, but not limited to, positron emissiontomography and computed tomography (PET-CT), single-photon emissioncomputed tomography (SPECT-CT), magnetic resonance spectroscopy (MR),X-ray computed tomography (CT), and molecular imaging.

The present invention provides methods for treating dysplasia of thedigestive system in a subject comprising administering a trefoil familymolecule. Dysplasia is a condition where there is a morphologicallyidentifiable local tissue abnormality at a given site. Dysplasia canhave characteristics including, but not limited to, increased cellnumber, nuclear abnormalities, and cellular differentiationabnormalities, compared to normal cells. A dysplasia can precede thedevelopment of any neoplasm, benign or malignant.

The present invention provides methods for decreasing cell proliferationin a subject comprising administering a trefoil family molecule. In oneembodiment, the cells are myeloid-derived suppressor cells (alsoreferred to throughout as “MDSC”). In another embodiment themyeloid-derived suppressor cells are tumor associated. In anotherembodiment, the myeloid-derived suppressor cell is a myeloid-derivedsuppressor cell associated with a tumor. For example, the tumor can beany solid tumor associated with myeloid-derived suppressor cells. Atumor is a growth of tissue forming an abnormal mass, and can be benign,pre-malignant, or malignant. In one embodiment, the tumor is a breasttumor. In another embodiment, the tumor is a prostate tumor. In anotherembodiment, the tumor is a lung tumor. In a further embodiment, thetumor is a skin tumor. In one embodiment the tumor can be a tumor of thedigestive system. In one embodiment, the tumor of the digestive systemincludes, but is not limited to, a mouth tumor, a pharynx tumor, anesophageal tumor, a stomach tumor, a small intestine tumor, a largeintestine tumor, a colon tumor, a rectal tumor, an anal tumor, a livertumor, a pancreatic tumor, and a gall bladder tumor.

In a further embodiment MDSC express a surface marker. In yet anotherembodiment MDSC do not express a surface marker. In one embodiment, MDSCexpress the surface marker Grl1, CD11b, or a combination thereof. In oneembodiment, MDSC express the surface marker CD14, CD15, CD33, or acombination thereof. In another embodiment, MDSC do not express thesurface marker HLA-DR. In a further embodiment, MDSC express the surfacemarker CD14, CD15, CD33, and do not express the surface marker HLA-DR,or a combination thereof.

MDSC are a heterogeneous population of early myeloid progenitors, suchas immature granulocytes, macrophages, and dendritic cells. As usedherein “MDSC” includes both M-MDSC (monocytic-MDSC) or G-MDSC(granulocytic-MDSC), or a combination thereof. MDSC may or may notexpress surface markers. For example, in mice, MDSC can express CD11b,Gr1, or a combination thereof. In mice, MDSC can express other surfacemarkers including, but not limited to Ly-6G, Ly-6C, CD49d, or acombination thereof. In one embodiment, mouse MDSC express CD11b, Gr1,and Ly-6G and do not express Ly-6C and CD49d. In another embodiment,mouse MDSC express CD80, CD11b, Grl1, Ly-6C and CD49d and do not expressLy-6G. In humans, MDSC can express CD14, CD15, CD33, or a combinationthereof. In humans, MDSC may not express HLA-DR. In one embodiment,human MDSC express CD14, CD33 and do not express HLA-DR. In anotherembodiment, human MDSC express CD15, CD33 and do not express HLA-DR. Inhumans, MDSC can express other surface markers including, but notlimited to, CD11b, CD124, S100A9, Stat, CD80, CD83, DC-Sign, SSC, or acombination thereof. In humans, MDSC may not express other surfacemarkers, including, but not limited to, Lin (Lin refers to Lineagemarkers specific for T and B cells). In one embodiment, MDSC can beassociated with tumors. In another embodiment MDSC can reside in thetumor microenvironment. The tumor microenvironment comprises the normalcells and molecules that surround a tumor or cancer cell.Characteristics of MDSC will be known to one of skill in the art, forfurther information the reader is referred to Lindau D. et al. 2013Immunology 138(2):105-115.

The present invention also provides a kit for treating a trefoil familymolecule disorder in a subject. A trefoil family molecule disordercomprises an inflammatory disease of the digestive system, a cancer ofthe digestive system, or a dysplasia of the digestive system. In oneembodiment, the kit for treating a trefoil family molecule disordercomprises a trefoil family molecule, to administer to a subject andinstructions of use.

The present invention also provide a method of determining the presenceof, or predisposition to, a trefoil family molecule disorder in asubject. A trefoil family molecule disorder comprises an inflammatorydisease of the digestive system, a cancer of the digestive system, or adysplasia of the digestive system. In one embodiment, the presence of,or predisposition to a trefoil family molecule disorder in a subject isdetermined by extracting a sample from a subject and detecting thepresence, absence or reduction of a trefoil family molecule in thesample, wherein absence, or reduction of the molecule indicates thepresence of, or predisposition to, a trefoil family molecule disorder.In a further embodiment, the method further comprises administering atrefoil family molecule to the subject where a trefoil family proteinwas not detected. In one embodiment the sample is digestive systemcancer cells. In one embodiment, a reduction of a trefoil familymolecule in the sample comprises detecting a lower amount of a trefoilfamily molecule in the sample than the amount of a trefoil familymolecule in a control sample. In one embodiment, the control sample isfrom a subject without a trefoil family molecule disorder. In anotherembodiment, the control sample are not cancer cells. In one embodiment,the trefoil family molecule is detected by incubating the sample with anagent that binds to a trefoil family molecule. In a further embodiment,the agent is an antibody to a trefoil family molecule.

The present invention also provides a diagnostic kit for determining thepresence of, or predisposition to, a trefoil family protein disorder,the kit comprising an agent that binds to a trefoil family molecule, andinstructions for use. A trefoil family molecule disorder comprises aninflammatory disease of the digestive system, a cancer of the digestivesystem, or a dysplasia of the digestive system. In one embodiment, theagent is an antibody to a trefoil family molecule.

In one embodiment, the subject is an animal. In another embodiment, thesubject is an animal that has or is diagnosed with a disease of thedigestive system. In one embodiment, the subject is a human. In otherembodiments, the subject is a mammal. In one embodiment, the subject isa dog. In another embodiment, the subject is a cat. In some embodiments,the subject is a rodent, such as a mouse or a rat. In some embodiments,the subject is a cow, pig, sheep, goat, cat, horse, dog, and/or anyother species of animal used as livestock or kept as pets.

Trefoil Family Molecules

As used herein, a “trefoil family molecule” refers to a trefoil familyprotein, or a fragment thereof. A “trefoil family molecule” can alsorefer to a nucleic acid (including, for example, genomic DNA,complementary DNA (cDNA), synthetic DNA, as well as any form ofcorresponding RNA) which encodes a polypeptide corresponding to atrefoil family protein, or fragment thereof. For example, a trefoilfamily molecule can include TFF1 (e.g., comprising the amino acidsequence shown in SEQ ID NO: 1, or comprising the nucleic acid sequenceshown in SEQ ID NO: 2), TFF2 (e.g., comprising the amino acid sequenceshown in SEQ ID NO: 3, or comprising the nucleic acid sequence shown inSEQ ID NO: 4), or TFF3 (e.g., comprising the amino acid sequence shownin SEQ ID NO: 5, or comprising the nucleic acid sequence shown in SEQ IDNO: 6). For example, a trefoil family molecule can be encoded by arecombinant nucleic acid encoding a trefoil family protein, or fragmentthereof. The trefoil family molecules of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that encodes a trefoilfamily molecule can be obtained by screening DNA libraries, or byamplification from a natural source. A trefoil family molecule caninclude a fragment or portion of a trefoil family protein. A trefoilfamily molecule can include a variant of the above described examples,such as a fragment thereof. Such a variant can comprise anaturally-occurring variant due to allelic variations betweenindividuals (e.g., polymorphisms), mutated alleles, or alternativesplicing forms. In one embodiment, a trefoil family molecule is encodedby a nucleic acid variant of the nucleic acid having the sequence shownin SEQ ID NOS: 2, 4, or 6 wherein the variant has a nucleotide sequenceidentity to SEQ ID NOS:2, 4, or 6 of at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99%. In another embodiment, a variantof the trefoil family protein comprises a protein or polypeptide encodedby a trefoil family nucleic acid sequence, such as the sequence shown inSEQ ID NOS: 2, 4, or 6. A trefoil family molecule can also include atrefoil family protein, or fragment thereof, that is modified by theaddition of a carboxy-terminal peptide (CTP) domain for increasedstability.

The nucleic acid can be any type of nucleic acid, including genomic DNA,complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well asany form of corresponding RNA. For example, a nucleic acid encoding atrefoil family protein can comprise a recombinant nucleic acid encodingsuch a protein. The nucleic acid can be a non-naturally occurringnucleic acid created artificially (such as by assembling, cutting,ligating or amplifying sequences). It can be double-stranded orsingle-stranded.

The invention further provides for nucleic acids that are complementaryto a trefoil family molecule. Complementary nucleic acids can hybridizeto the nucleic acid sequence described above under stringenthybridization conditions. Non-limiting examples of stringenthybridization conditions include temperatures above 30° C., above 35°C., in excess of 42° C., and/or salinity of less than about 500 mM, orless than 200 mM. Hybridization conditions can be adjusted by theskilled artisan via modifying the temperature, salinity and/or theconcentration of other reagents such as SDS or SSC.

According to the invention, protein variants can include amino acidsequence modifications. For example, amino acid sequence modificationsfall into one or more of three classes: substitutional, insertional ordeletional variants. Insertions can include amino and/or carboxylterminal fusions as well as intrasequence insertions of single ormultiple amino acid residues. Insertions ordinarily will be smallerinsertions than those of amino or carboxyl terminal fusions, forexample, on the order of one to four residues. Deletions arecharacterized by the removal of one or more amino acid residues from theprotein sequence. These variants ordinarily are prepared bysite-specific mutagenesis of nucleotides in the DNA encoding theprotein, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. In one embodiment, atrefoil family molecule can be modified with an amino acid sequenceinserted as a carboxyl terminal fusion. For example, carboxyl terminalfusions may be used to increase the stability of a trefoil familymolecule.

In one embodiment, a trefoil family molecule comprises a protein orpolypeptide encoded by a nucleic acid sequence encoding a trefoil familyprotein, such as the sequences shown in SEQ ID NOS: 2, 4, or 6. Inanother embodiment, the polypeptide can be modified, such as byglycosylations and/or acetylations and/or chemical reaction or coupling,and can contain one or several non-natural or synthetic amino acids. Anexample of a trefoil family molecule is the polypeptide having the aminoacid sequence shown in SEQ ID NOS: 1, 3, or 5. Such variants can includethose having at least from about 46% to about 50% identity to SEQ IDNOS: 1, 3, or 5 or having at least from about 50.1% to about 55%identity to SEQ ID NOS: 1, 3, or 5, or having at least from about 55.1%to about 60% identity to SEQ ID NOS: 1, 3, or 5, or having from at leastabout 60.1% to about 65% identity to SEQ ID NOS: 1, 3, or 5, or havingfrom about 65.1% to about 70% identity to SEQ ID NOS: 1, 3, or 5, orhaving at least from about 70.1% to about 75% identity to SEQ ID NOS: 1,3, or 5, or having at least from about 75.1% to about 80% identity toSEQ ID NOS: 1, 3, or 5, or having at least from about 80.1% to about 85%identity to SEQ ID NOS: 1, 3, or 5, or having at least from about 85.1%to about 90% identity to SEQ ID NOS: 1, 3, or 5, or having at least fromabout 90.1% to about 95% identity to SEQ ID NOS: 1, 3, or 5, or havingat least from about 95.1% to about 97% identity to SEQ ID NOS: 1, 3, or5, or having at least from about 97.1% to about 99% identity to SEQ IDNOS: 1, 3, or 5. In another embodiment, a trefoil family molecule can bea fragment of a trefoil family protein.

In one embodiment, a trefoil family molecule, according to the methodsdescribed herein can be administered to a subject as a recombinantprotein. In another embodiment, a trefoil family molecule, can beadministered to a subject as a modified recombinant protein. Forexample, a trefoil family protein, or fragment thereof, can be modifiedby the addition of a carboxy-terminal peptide (CTP) domain for increasedstability. In a further embodiment, a trefoil family molecule, accordingto the methods described herein can be administered to a subject bydelivery of a nucleic acid encoding a trefoil family protein, orfragment thereof. For example, nucleic acids can be delivered to asubject using a viral vector.

Polypeptides can be susceptible to denaturation or enzymatic degradationin the blood, liver or kidney. Accordingly, polypeptides can be unstableand have short biological half-lives. Polypeptides can be modified toincrease their stability, for example, a fusion protein can be generatedfor increased stability. In one embodiment, an isolated polypeptide cancomprise a carboxy-terminal peptide (CTP) domain fused to a trefoilfamily molecule. The addition of the CTP domain to a trefoil familymolecule can be used to stabilize the trefoil family molecule and causea longer biological half-life to the polypeptides in circulation. In oneembodiment, the CTP comprises the C-terminal domain of the beta subunitof the human chorionic gonadotrophin (hCG).

The term “biological half-life” is the time required for the activity ofa substance taken into the body to lose one half its initialpharmacologic, physiologic, or biologic activity.

In one embodiment, a trefoil family molecule of the present inventioncomprises an isolated polypeptide comprising a carboxy-terminal peptide(CTP) domain fused to a trefoil family molecule. In one embodiment,fusing a CTP domain to a trefoil family molecule (for example, TFF1,TFF2, or TFF3) can result in increased glycosylation and/or proteinstability. In some embodiments, one CTP domain is added to theN-terminus of a trefoil family molecule. In other embodiments, two CTPdomains are added to the N-terminus of a trefoil family molecule. Infurther embodiments, three CTP domains are added to the N-terminus of atrefoil family molecule. In some embodiments, one CTP domain is added tothe C-terminus of a trefoil family molecule. In other embodiments, twoCTP domains are added to the C-terminus of a trefoil family molecule. Infurther embodiments, three CTP domains are added to the C-terminus of atrefoil family molecule. In some embodiments, at least one CTP domain isadded to the N-terminus and/or C-terminus of a trefoil family molecule.In other embodiments, at least two CTP domains are added to theN-terminus and/or C-terminus of a trefoil family molecule. In furtherembodiments, at least three CTP domains are added to the N-terminusand/or C-terminus of a trefoil family molecule. In some embodiments, theCTP domains are added in tandem.

In one embodiment, a trefoil family molecule of the present inventioncomprises an isolated polypeptide comprising a Fc domain fused to atrefoil family molecule. A Fc domain is the fragment crystallizableregion of an antibody. In one embodiment, fusing a Fc domain to atrefoil family molecule (for example, TFF1, TFF2, or TFF3) can result indimerization, and/or protein stability, and/or increased proteinactivity, and/or improved protein purification. In some embodiments, oneFc domain is added to the N-terminus of a trefoil family molecule. Inother embodiments, two Fc domains are added to the N-terminus of atrefoil family molecule. In further embodiments, three Fc domains areadded to the N-terminus of a trefoil family molecule. In someembodiments, one Fc domain is added to the C-terminus of a trefoilfamily molecule. In other embodiments, two Fc domains are added to theC-terminus of a trefoil family molecule. In further embodiments, threeFc domains are added to the C-terminus of a trefoil family molecule. Insome embodiments, at least one Fc domain is added to the N-terminusand/or C-terminus of a trefoil family molecule. In other embodiments, atleast two Fc domains are added to the N-terminus and/or C-terminus of atrefoil family molecule. In further embodiments, at least three Fcdomains are added to the N-terminus and/or C-terminus of a trefoilfamily molecule. In some embodiments, the Fc domains are added intandem.

In one embodiment, a trefoil family molecule of the present inventioncomprises an isolated polypeptide comprising a CTP domain and a Fcdomain fused to a trefoil family molecule. In one embodiment, fusing aCTP domain and a Fc domain to a trefoil family molecule (for example,TFF1, TFF2, or TFF3) can result in dimerization, and/or proteinstability, and/or increased protein activity, and/or improved proteinpurification. In some embodiments, a CTP domain and a Fc domain areadded to the N-terminus of a trefoil family molecule. In someembodiments, a CTP domain and a Fc domain are added to the C-terminus ofa trefoil family molecule. In some embodiments, at least one Fc domainis added to the N-terminus and/or C-terminus of a trefoil familymolecule and at least one CTP domain is added to the N-terminus and/orC-terminus of a trefoil family molecule. In other embodiments, at leastone Fc domain is added to the N-terminus and/or C-terminus of a trefoilfamily molecule and at least two CTP domains are added to the N-terminusand/or C-terminus of a trefoil family molecule. In further embodiments,at least one Fc domain are added to the N-terminus and/or C-terminus ofa trefoil family molecule and at least three CTP domain is added to theN-terminus and/or C-terminus of a trefoil family molecule. In someembodiments, the Fc domains and CTP domains are added in tandem and canbe in any order.

SEQ ID NO: 19 depicts the amino acid sequence of a CTP domain:

GSPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ

SEQ ID NO: 20 depicts the nucleic acid sequence encoding a CTP domain:

ggatcaccacgcttccaggactcctcttcctcaaaggcccctcctcctagccttccaagcccatcccgactcccggggccctcggacactccgatcctcc cacaataa

SEQ ID NO: 21 depicts the amino acid sequence of a Fc domain:

  1 MWGWKCLLFW AVLVTATLCT ARPAPTLPEQ AQQSTRADLG PGEPKSCDKT HTCPPCPAPE 61 LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE121 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP181 SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD241 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

SEQ ID NO: 22 depicts the nucleic acid sequence encoding a Fc domain:

  1 atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact 61 gccaggccag ccccaacctt gcccgaacaa gctcagcagt cgacgcgcgc agatctgggc121 ccgggcgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa181 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc241 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc301 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag361 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg421 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag481 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca541 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat601 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc661 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac721 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac781 aaccactaca cgcagaagag cctctccctg tctccgggta aa

SEQ ID NO: 23 depicts the amino acid sequence of a FcCTP where the CTPdomain is underlined and bold:

  1 MWGWKCLLFW AVLVTATLCT ARPAPTLPEQ AQQSTRADLG PGEPKSCDKT HTCPPCPAPE 61 LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE121 EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP181 SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD241 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK GSPRFQ DSSSSKAPPP SLPSPSRLPG301  PSDTPILPQ

SEQ ID NO: 24 depicts the nucleic acid sequence encoding a FcCTP wherethe CTP domain is underlined and bold:

  1 atgtggggct ggaagtgcct cctcttctgg gctgtgctgg tcacagccac tctctgcact 61 gccaggccag ccccaacctt gcccgaacaa gctcagcagt cgacgcgcgc agatctgggc121 ccgggcgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa181 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc241 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc301 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag361 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg421 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag481 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca541 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat601 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc661 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac721 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac781 aaccactaca cgcagaagag cctctccctg tctccgggta aa ggatcacc acgcttccag841  gactcctctt cctcaaaggc ccctcctcct agccttccaa gcccatcccg actcccgggg901  ccctcggaca ctccgatcct cccacaataa

The invention provides for a nucleic acid encoding a trefoil familyprotein, or fragment thereof, such as a TFF1 molecule, a TFF2 molecule,or a TFF3 molecule.

For example, the polypeptide sequence of human TFF1 is depicted in SEQID NO: 1. The nucleotide sequence of human TFF1 is shown in SEQ ID NO:2. Sequence information related to TFF1 is accessible in publicdatabases by GenBank Accession numbers NP_(—)003216.1 (protein) and NM003225.2 (nucleic acid).

SEQ ID NO: 1 is the human wild type amino acid sequence corresponding toTFF1 (residues 1-84):

 1 MATMENKVIC ALVLVSMLAL GTLAEAQTET CTVAPRERQN CGFPGVTPSQ CANKGCCFDD61 TVRGVPWCFY PNTIDVPPEE ECEF

SEQ ID NO: 2 is the human wild type nucleotide sequence corresponding toTFF1 (nucleotides 1-508), wherein the underscored bolded “ATG” denotesthe beginning of the open reading frame:

  1 atccctgact cggggtcgcc tttggagcag agaggaggca  atg gccacca tggagaacaa 61 ggtgatctgc gccctggtcc tggtgtccat gctggccctc ggcaccctgg ccgaggccca121 gacagagacg tgtacagtgg ccccccgtga aagacagaat tgtggttttc ctggtgtcac181 gccctcccag tgtgcaaata agggctgctg tttcgacgac accgttcgtg gggtcccctg241 gtgcttctat cctaatacca tcgacgtccc tccagaagag gagtgtgaat tttagacact301 tctgcaggga tctgcctgca tcctgacgcg gtgccgtccc cagcacggtg attagtccca361 gagctcggct gccacctcca ccggacacct cagacacgct tctgcagctg tgcctcggct421 cacaacacag attgactgct ctgactttga ctactcaaaa ttggcctaaa aattaaaaga481 gatcgatatt aaaaaaaaaa aaaaaaaa

For example, the polypeptide sequence of human TFF2 is depicted in SEQID NO: 3. The nucleotide sequence of human TFF2 is shown in SEQ ID NO:4. Sequence information related to TFF2 is accessible in publicdatabases by GenBank Accession numbers NP_(—)005414.1 (protein) and NM005423.4 (nucleic acid).

SEQ ID NO: 3 is the human wild type amino acid sequence corresponding toTFF2 (residues 1-129):

  1 MGRRDAQLLA ALLVLGLCAL AGSEKPSPCQ CSRLSPHNRT NCGFPGITSD QCFDNGCCFD 61 SSVTGVPWCF HPLPKQESDQ CVMEVSDRRN CGYPGISPEE CASRKCCFSN FIFEVPWCFF121 PKSVEDCHY

SEQ ID NO: 4 is the human wild type nucleotide sequence corresponding toTFF2 (nucleotides 1-717), wherein the underscored bolded “ATG” denotesthe beginning of the open reading frame:

  1 cacggtggaa gggctggggc cacggggcag agaagaaagg ttatctctgc ttgttggaca 61 aacagagggg agattataaa acatacccgg cagtggacac catgcattct gcaagccacc121 ctggggtgca gctgagctag ac atg ggacg gcgagacgcc cagctcctgg cagcgctcct181 cgtcctgggg ctatgtgccc tggcggggag tgagaaaccc tccccctgcc agtgctccag241 gctgagcccc cataacagga cgaactgcgg cttccctgga atcaccagtg accagtgttt301 tgacaatgga tgctgtttcg actccagtgt cactggggtc ccctggtgtt tccaccccct361 cccaaagcaa gagtcggatc agtgcgtcat ggaggtctca gaccgaagaa actgtggcta421 cccgggcatc agccccgagg aatgcgcctc tcggaagtgc tgcttctcca acttcatctt481 tgaagtgccc tggtgcttct tcccgaagtc tgtggaagac tgccattact aagagaggct541 ggttccagag gatgcatctg gctcaccggg tgttccgaaa ccaaagaaga aacttcgcct601 tatcagcttc atacttcatg aaatcctggg ttttcttaac catcttttcc tcattttcaa661 tggtttaaca tataatttct ttaaataaaa cccttaaaat ctgctaaaaa aaaaaaa

For example, the polypeptide sequence of human TFF3 is depicted in SEQID NO: 5. The nucleotide sequence of human TFF3 is shown in SEQ ID NO:6. Sequence information related to TFF3 is accessible in publicdatabases by GenBank Accession numbers NP_(—)003217.3 (protein) and NM003226.3 (nucleic acid).

SEQ ID NO: 5 is the human wild type amino acid sequence corresponding toTFF3 (residues 1-94):

 1 MKRVLSCVPE PTVVMAARAL CMLGLVLALL SSSSAEEYVG LSANQCAVPA KDRVDCGYPH61 VTPKECNNRG CCFDSRIPGV PWCFKPLQEA ECTF

SEQ ID NO: 6 is the human wild type nucleotide sequence corresponding toTFF3 (nucleotides 1-1054), wherein the underscored bolded “ATG” denotesthe beginning of the open reading frame:

   1 gccaaaacag tgggggctga actgacctct cccctttggg agagaaaaac tgtctgggag  61 cttgacaaag gcatgcagga gagaacagga gcagccacag ccaggaggga gagccttccc 121 caagcaaaca atccagagca gctgtgcaaa caacggtgca taaatgaggc ctcctggacc 181  atg aagcgag tcctgagctg cgtcccggag cccacggtgg tcatggctgc cagagcgctc 241 tgcatgctgg ggctggtcct ggccttgctg tcctccagct ctgctgagga gtacgtgggc 301 ctgtctgcaa accagtgtgc cgtgccagcc aaggacaggg tggactgcgg ctacccccat 361 gtcaccccca aggagtgcaa caaccggggc tgctgctttg actccaggat ccctggagtg 421 ccttggtgtt tcaagcccct gcaggaagca gaatgcacct tctgaggcac ctccagctgc 481 ccccggccgg gggatgcgag gctcggagca cccttgcccg gctgtgattg ctgccaggca 541 ctgttcatct cagcttttct gtccctttgc tcccggcaag cgcttctgct gaaagttcat 601 atctggagcc tgatgtctta acgaataaag gtcccatgct ccacccgagg acagttcttc 661 gtgcctgaga ctttctgagg ttgtgcttta tttctgctgc gtcgtgggag agggcgggag 721 ggtgtcaggg gagagtctgc ccaggcctca agggcaggaa aagactccct aaggagctgc 781 agtgcatgca aggatatttt gaatccagac tggcacccac gtcacaggaa agcctaggaa 841 cactgtaagt gccgcttcct cgggaaagca gaaaaaatac atttcaggta gaagttttca 901 aaaatcacaa gtctttcttg gtgaagacag caagccaata aaactgtctt ccaaagtggt 961 cctttatttc acaaccactc tcgctactgt tcaatacttg tactattcct gggttttgtt1021 tctttgtaca gtaaacatta tgaacaaaca ggca

A trefoil family molecule can also encompass ortholog genes, which aregenes conserved among different biological species such as humans, dogs,cats, mice, and rats, that encode proteins (for example, homologs(including splice variants), mutants, and derivatives) havingbiologically equivalent functions as the human-derived protein.Orthologs of a trefoil family protein include any mammalian orthologinclusive of the ortholog in humans and other primates, experimentalmammals (such as mice, rats, hamsters and guinea pigs), mammals ofcommercial significance (such as horses, cows, camels, pigs and sheep),and also companion mammals (such as domestic animals, e.g., rabbits,ferrets, dogs, and cats). A trefoil family molecule can comprise aprotein encoded by a nucleic acid sequence homologous to the humannucleic acid, wherein the nucleic acid is found in a different speciesand wherein that homolog encodes a protein similar to a trefoil familyprotein.

The invention utilizes conventional molecular biology, microbiology, andrecombinant DNA techniques available to one of ordinary skill in theart. Such techniques are well known to the skilled worker and areexplained fully in the literature. See, e.g., Maniatis, Fritsch &Sambrook, “DNA Cloning: A Practical Approach,” Volumes I and II (D. N.Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “ImmobilizedCells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” (2001).

One skilled in the art can obtain a trefoil family molecule, (e.g.,TFF1, TFF2, or TFF3) in several ways, which include, but are not limitedto, isolating the protein via biochemical means or expressing anucleotide sequence encoding the protein of interest by geneticengineering methods.

The invention provides for a trefoil family molecule that are encoded bynucleotide sequences. The trefoil family molecule can be a polypeptideencoded by a nucleic acid (including genomic DNA, complementary DNA(cDNA), synthetic DNA, as well as any form of corresponding RNA). Forexample, a trefoil family molecule can be encoded by a recombinantnucleic acid encoding a human trefoil family protein, or fragmentthereof. The trefoil family molecules of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that encodes a trefoilfamily molecule can be obtained by screening DNA libraries, or byamplification from a natural source. The trefoil family molecule of theinvention can be produced via recombinant DNA technology and suchrecombinant nucleic acids can be prepared by conventional techniques,including chemical synthesis, genetic engineering, enzymatic techniques,or a combination thereof. A trefoil family molecule of this inventioncan also encompasses variants of the human trefoil family proteins. Thevariants can comprise naturally-occurring variants due to allelicvariations between individuals (e.g., polymorphisms), mutated alleles,or alternative splicing forms.

In one embodiment, a fragment of a nucleic acid sequence that comprisesa trefoil family molecule (such as, e.g., TFF1, TFF2, or TFF3) canencompass any portion of at least about 8 consecutive nucleotides of SEQID NO: 2, 4, or 6. In one embodiment, the fragment can comprise at leastabout 10 nucleotides, at least about 15 nucleotides, at least about 20nucleotides, or at least about 30 nucleotides of SEQ ID NO: 2, 4, or 6.Fragments include all possible nucleotide lengths between about 8 andabout 100 nucleotides, for example, lengths between about 15 and about100 nucleotides, or between about 20 and about 100 nucleotides.

A trefoil family molecule, can be a fragment of a trefoil familyprotein, such as, e.g., TFF1, TFF2, or TFF3. For example, the trefoilfamily protein fragment can encompass any portion of at least about 8consecutive amino acids of SEQ ID NO: 1, 3, or 5. The fragment cancomprise at least about 10 consecutive amino acids, at least about 20consecutive amino acids, at least about 30 consecutive amino acids, atleast about 40 consecutive amino acids, a least about 50 consecutiveamino acids, at least about 60 consecutive amino acids, at least about70 consecutive amino acids, at least about 80 consecutive amino acids,at least about 90 consecutive amino acids, at least about 100consecutive amino acids, at least about 110 consecutive amino acids, orat least about 120 consecutive amino acids of SEQ ID NOS: 1, 3, or 5.Fragments include all possible amino acid lengths between about 8 and 80about amino acids, for example, lengths between about 10 and about 80amino acids, between about 15 and about 80 amino acids, between about 20and about 80 amino acids, between about 35 and about 80 amino acids,between about 40 and about 80 amino acids, between about 50 and about 80amino acids, or between about 70 and about 80 amino acids.

Recombinant Proteins

One skilled in the art understands that polypeptides (for example TFF1,TFF2, TFF3, and the like) can be obtained in several ways, which includebut are not limited to, expressing a nucleotide sequence encoding theprotein of interest, or fragment thereof, by genetic engineeringmethods.

In one embodiment, the nucleic acid is expressed in an expressioncassette, for example, to achieve overexpression in a cell. The nucleicacids of the invention can be an RNA, cDNA, cDNA-like, or a DNA ofinterest in an expressible format, such as an expression cassette, whichcan be expressed from the natural promoter or an entirely heterologouspromoter. The nucleic acid of interest can encode a protein, and may ormay not include introns. Any recombinant expression system can be used,including, but not limited to, bacterial, mammalian, yeast, insect, orplant cell expression systems.

Host cells transformed with a nucleic acid sequence encoding a trefoilfamily molecule (such as, e.g., TFF1, TFF2, or TFF3), can be culturedunder conditions suitable for the expression and recovery of the proteinfrom cell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. Expression vectors containing a nucleic acid sequenceencoding a trefoil family molecule can be designed to contain signalsequences which direct secretion of soluble polypeptide moleculesencoded by a trefoil family molecule (such as, e.g., TFF1, TFF2, orTFF3), through a prokaryotic or eukaryotic cell membrane.

Nucleic acid sequences comprising a trefoil family molecule (such as,e.g., TFF1, TFF2, or TFF3) that encode a polypeptide can be synthesized,in whole or in part, using chemical methods known in the art.Alternatively, a trefoil family molecule can be produced using chemicalmethods to synthesize its amino acid sequence, such as by direct peptidesynthesis using solid-phase techniques. Protein synthesis can either beperformed using manual techniques or by automation. Automated synthesiscan be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Optionally, fragments of a trefoil familymolecule can be separately synthesized and combined using chemicalmethods to produce a full-length molecule.

A synthetic peptide can be substantially purified via high performanceliquid chromatography (HPLC). The composition of a synthetic a trefoilfamily molecule can be confirmed by amino acid analysis or sequencing.Additionally, any portion of an amino acid sequence comprising a proteinencoded by a trefoil family molecule (e.g., TFF1, TFF2, or TFF3) can bealtered during direct synthesis and/or combined using chemical methodswith sequences from other proteins to produce a variant polypeptide or afusion protein.

The invention further encompasses methods for using a protein orpolypeptide encoded by a nucleic acid sequence of a trefoil familymolecule, such as the sequences shown in SEQ ID NOS: 1, 3, or 5. Inanother embodiment, the polypeptide can be modified, such as byglycosylations and/or acetylations and/or chemical reaction or coupling,and can contain one or several non-natural or synthetic amino acids. Anexample of a trefoil family molecule has the amino acid sequence shownin either SEQ ID NO: 1, 3, or 5. In certain embodiments, the inventionencompasses variants of a human protein encoded by a trefoil familymolecule (such as, e.g., TFF1, TFF2, and TFF3).

Expression Systems

Bacterial Expression Systems.

One skilled in the art understands that expression of desired proteinproducts in prokaryotes is most often carried out in E. coli withvectors that contain constitutive or inducible promoters. Somenon-limiting examples of bacterial cells for transformation include thebacterial cell line E. coli strains DH5a or MC1061/p3 (Invitrogen Corp.,San Diego, Calif.), which can be transformed using standard procedurespracticed in the art, and colonies can then be screened for theappropriate plasmid expression. In bacterial systems, a number ofexpression vectors can be selected. Non-limiting examples of suchvectors include multifunctional E. coli cloning and expression vectorssuch as BLUESCRIPT (Stratagene). Some E. coli expression vectors (alsoknown in the art as fusion-vectors) are designed to add a number ofamino acid residues, usually to the N-terminus of the expressedrecombinant protein. Such fusion vectors can serve three functions: 1)to increase the solubility of the desired recombinant protein; 2) toincrease expression of the recombinant protein of interest; and 3) toaid in recombinant protein purification by acting as a ligand inaffinity purification. In some instances, vectors, which direct theexpression of high levels of fusion protein products that are readilypurified, may also be used. Some non-limiting examples of fusionexpression vectors include pGEX, which fuse glutathione S-tranferase(GST) to desired protein; pcDNA 3.1/V5-His A B & C (Invitrogen Corp,Carlsbad, Calif.) which fuse 6×-His to the recombinant proteins ofinterest; pMAL (New England Biolabs, MA) which fuse maltose E bindingprotein to the target recombinant protein; the E. coli expression vectorpUR278 (Ruther et al., (1983) EMBO 12:1791), wherein the coding sequencemay be ligated individually into the vector in frame with the lac Zcoding region in order to generate a fusion protein; and pIN vectors(Inouye et al., (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke etal., (1989) J. Biol. Chem. 24:5503-5509. Fusion proteins generated bythe likes of the above-mentioned vectors are generally soluble and canbe purified easily from lysed cells via adsorption and binding of thefusion protein to an affinity matrix. For example, fusion proteins canbe purified from lysed cells via adsorption and binding to a matrix ofglutathione agarose beads subsequently followed by elution in thepresence of free glutathione. For example, the pGEX vectors are designedto include thrombin or factor Xa protease cleavage sites so that thecloned target can be released from the GST moiety.

Plant, Insect, and Yeast Expression Systems.

Other suitable cell lines, in addition to microorganisms such asbacteria (e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining coding sequences for a trefoil family molecule mayalternatively be used to produce the molecule of interest. Anon-limiting example includes plant cell systems infected withrecombinant virus expression vectors (for example, tobacco mosaic virus,TMV; cauliflower mosaic virus, CaMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing codingsequences for a trefoil family molecule. If plant expression vectors areused, the expression of sequences encoding a trefoil family molecule canbe driven by any of a number of promoters. For example, viral promoterssuch as the 35S and 19S promoters of CaMV can be used alone or incombination with the omega leader sequence from tobacco mosaic virusTMV. Alternatively, plant promoters such as the small subunit of RUBISCOor heat shock promoters, can be used. These constructs can be introducedinto plant cells by direct DNA transformation or by pathogen-mediatedtransfection.

In another embodiment, an insect system also can be used to express atrefoil family molecule. For example, in one such system Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. Sequences encoding a trefoil family molecule can be cloned intoa non-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe nucleic acid sequences of a trefoil family molecule will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein. The recombinant viruses can then be used to infect S.frugiperda cells or Trichoplusia larvae in which a trefoil familymolecule can be expressed.

In another embodiment, a yeast (for example, Saccharomyces sp., Pichiasp.) system also can be used to express a trefoil family molecule. Yeastcan be transformed with recombinant yeast expression vectors containingcoding sequences for a trefoil family molecule.

Mammalian Expression Systems.

Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like)can also contain an expression vector (for example, one that harbors anucleotide sequence encoding a trefoil family molecule) for expressionof a desired product. Expression vectors containing such a nucleic acidsequence linked to at least one regulatory sequence in a manner thatallows expression of the nucleotide sequence in a host cell can beintroduced via methods known in the art. A number of viral-basedexpression systems can be used to express a trefoil family molecule inmammalian host cells. The vector can be a recombinant DNA or RNA vector,and includes DNA plasmids or viral vectors. For example, if anadenovirus is used as an expression vector, sequences encoding a trefoilfamily molecule can be ligated into an adenovirustranscription/translation complex comprising the late promoter andtripartite leader sequence. Insertion into a non-essential E1 or E3region of the viral genome can be used to obtain a viable virus which iscapable of expressing a trefoil family molecule in infected host cells.Transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,can also be used to increase expression in mammalian host cells. Inaddition, viral vectors can be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, lentivirus oralphavirus.

Regulatory sequences are well known in the art, and can be selected todirect the expression of a protein or polypeptide of interest (such as atrefoil family molecule) in an appropriate host cell as described inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Non-limiting examples of regulatorysequences include: polyadenylation signals, promoters (such as CMV, ASV,SV40, or other viral promoters such as those derived from bovinepapilloma, polyoma, and Adenovirus 2 viruses (Fiers, et al., 1973,Nature 273:113; Hager G L, et al., Curr Opin Genet Dev, 2002,12(2):137-41) enhancers, and other expression control elements.Practitioners in the art understand that designing an expression vectorcan depend on factors, such as the choice of host cell to be transfectedand/or the type and/or amount of desired protein to be expressed.

Enhancer regions, which are those sequences found upstream or downstreamof the promoter region in non-coding DNA regions, are also known in theart to be important in optimizing expression. If needed, origins ofreplication from viral sources can be employed, such as if a prokaryotichost is utilized for introduction of plasmid DNA. However, in eukaryoticorganisms, chromosome integration is a common mechanism for DNAreplication.

For stable transfection of mammalian cells, a small fraction of cellscan integrate introduced DNA into their genomes. The expression vectorand transfection method utilized can be factors that contribute to asuccessful integration event. For stable amplification and expression ofa desired protein, a vector containing DNA encoding a protein ofinterest (for example, a trefoil family molecule) is stably integratedinto the genome of eukaryotic cells (for example mammalian cells, suchas HEK293 cells), resulting in the stable expression of transfectedgenes. An exogenous nucleic acid sequence can be introduced into a cell(such as a mammalian cell, either a primary or secondary cell) byhomologous recombination as disclosed in U.S. Pat. No. 5,641,670, thecontents of which are herein incorporated by reference.

A gene that encodes a selectable marker (for example, resistance toantibiotics or drugs, such as ampicillin, neomycin, G418, andhygromycin) can be introduced into host cells along with the gene ofinterest in order to identify and select clones that stably express agene encoding a protein of interest. The gene encoding a selectablemarker can be introduced into a host cell on the same plasmid as thegene of interest or can be introduced on a separate plasmid. Cellscontaining the gene of interest can be identified by drug selectionwherein cells that have incorporated the selectable marker gene willsurvive in the presence of the drug. Cells that have not incorporatedthe gene for the selectable marker die. Surviving cells can then bescreened for the production of the desired protein molecule (forexample, a trefoil family molecule).

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed trefoilfamily molecule (such as, e.g., TFF1, TFF2, or TFF3) in the desiredfashion. Such modifications of the polypeptide include, but are notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation, and acylation. Post-translational processing which cleaves a“prepro” form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138),are available from the American Type Culture Collection (ATCC; 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

An exogenous nucleic acid can be introduced into a cell via a variety oftechniques known in the art, such as lipofection, microinjection,calcium phosphate or calcium chloride precipitation,DEAE-dextrin-mediated transfection, or electroporation. Electroporationis carried out at approximate voltage and capacitance to result in entryof the DNA construct(s) into cells of interest. Other methods used totransfect cells can also include modified calcium phosphateprecipitation, polybrene precipitation, liposome fusion, andreceptor-mediated gene delivery.

Animal or mammalian host cells capable of harboring, expressing, andsecreting large quantities of a trefoil family molecule of interest intothe culture medium for subsequent isolation and/or purification include,but are not limited to, Human Embryonic Kidney 293 cells (HEK-293) (ATCCCRL-1573); Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCCCCL-61), DG44 (Chasin et al., (1986) Som. Cell Molec. Genet, 12:555-556;Kolkekar et al., (1997) Biochemistry, 36:10901-10909; and WO 01/92337A2), dihydrofolate reductase negative CHO cells (CHO/dhfr−, Urlaub etal., (1980) Proc. Natl. Acad. Sci. U.S.A., 77:4216), and dp12. CHO cells(U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40(COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g.,293 cells, or 293 cells subcloned for growth in suspension culture,Graham et al., (1977) J. Gen. Virol., 36:59); baby hamster kidney cells(BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); Africangreen monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81);mouse sertoli cells (TM4; Mather (1980) Biol. Reprod., 23:243-251);human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells(MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); humanhepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562,ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells(Mather (1982) Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells.A cell line transformed to produce a trefoil family molecule can also bean immortalized mammalian cell line of lymphoid origin, which includebut are not limited to, a myeloma, hybridoma, trioma or quadroma cellline. The cell line can also comprise a normal lymphoid cell, such as aB cell, which has been immortalized by transformation with a virus, suchas the Epstein Barr virus (such as a myeloma cell line or a derivativethereof).

A host cell strain, which modulates the expression of the insertedsequences, or modifies and processes the nucleic acid in a specificfashion desired also may be chosen. Such modifications (for example,glycosylation and other post-translational modifications) and processing(for example, cleavage) of protein products may be important for thefunction of the protein. Different host cell strains have characteristicand specific mechanisms for the post-translational processing andmodification of proteins and gene products. As such, appropriate hostsystems or cell lines can be chosen to ensure the correct modificationand processing of the foreign protein expressed, such as a trefoilfamily molecule. Thus, eukaryotic host cells possessing the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used.Non-limiting examples of mammalian host cells include HEK-293, 3T3,W138, BT483, Hs578T, CHO, VERY, BHK, Hela, COS, BT2O, T47D, NS0 (amurine myeloma cell line that does not endogenously produce anyimmunoglobulin chains), CRL7O3O, MDCK, 293, HTB2, and HsS78Bst cells.

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland W L, et al., J Immunol Methods, 1983,56(2): 221-234) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cellculturing conditions can vary according to the type of host cellselected. Commercially available medium can be utilized.

Cells suitable for culturing can contain introduced expression vectors,such as plasmids or viruses. The expression vector constructs can beintroduced via transformation, microinjection, transfection,lipofection, electroporation, or infection. The expression vectors cancontain coding sequences, or portions thereof, encoding the proteins forexpression and production. Expression vectors containing sequencesencoding the produced proteins and polypeptides, as well as theappropriate transcriptional and translational control elements, can begenerated using methods well known to and practiced by those skilled inthe art. These methods include synthetic techniques, in vitrorecombinant DNA techniques, and in vivo genetic recombination which aredescribed in J. Sambrook et al., 201, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. and in F. M.Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y.

Purification of Recombinant Proteins

A trefoil family molecule (such as, e.g., TFF1, TFF2, or TFF3) can bepurified from any human or non-human cell which expresses thepolypeptide, including those which have been transfected with expressionconstructs that express a trefoil family molecule. A purified trefoilfamily molecule (such as, e.g., TFF1, TFF2, or TFF3) can be separatedfrom other compounds which normally associate with the trefoil familymolecules, in the cell, such as certain proteins, carbohydrates, orlipids, using methods practiced in the art. For protein recovery,isolation and/or purification, the cell culture medium or cell lysate iscentrifuged to remove particulate cells and cell debris. The desiredpolypeptide molecule (for example, a trefoil family molecule) isisolated or purified away from contaminating soluble proteins andpolypeptides by suitable purification techniques. Non-limitingpurification methods for proteins include: size exclusionchromatography; affinity chromatography; ion exchange chromatography;ethanol precipitation; reverse phase HPLC; chromatography on a resin,such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g.,Sephadex G-75, Sepharose; protein A sepharose chromatography for removalof immunoglobulin contaminants; and the like. Other additives, such asprotease inhibitors (e.g., PMSF or proteinase K) can be used to inhibitproteolytic degradation during purification. Purification proceduresthat can select for carbohydrates can also be used, e.g., ion-exchangesoft gel chromatography, or HPLC using cation- or anion-exchange resins,in which the more acidic fraction(s) is/are collected.

Methods of Administration

Nucleic Acid Delivery Methods. The invention provides methods fortreating a disease of the digestive system in a subject, e.g., aninflammatory disease of the digestive system, or a digestive systemcancer. In one embodiment, the method can comprise administering to thesubject a trefoil family molecule (e.g, a trefoil family polypeptide ora trefoil family polynucleotide).

Various approaches can be carried out to restore the activity orfunction of a trefoil family molecule (such as, e.g., TFF1, TFF2, orTFF3) in a subject, such as those carrying an altered trefoil familygene locus. For example, supplying wild-type trefoil family genefunction (such as, e.g., TFF1, TFF2, TFF3) to such subjects can treatinflammatory diseases of the digestive system, treat a cancer of thedigestive system, treat dysplasia of the digestive system, decreasetumor growth, or decrease cell proliferation (e.g., myeloid-derivedsuppressor cell proliferation). Increasing a trefoil family geneexpression level or activity (such as, e.g., TFF1, TFF2, or TFF3) can beaccomplished through gene or protein therapy.

A nucleic acid encoding a trefoil family molecule can be introduced intothe cells of a subject. For example, the wild-type gene (or fragmentthereof) can also be introduced into the cells of the subject in needthereof using a vector as described herein. The vector can be a viralvector or a plasmid. The gene can also be introduced as naked DNA. Thegene can be provided so as to integrate into the genome of the recipienthost cells, or to remain extra-chromosomal. Integration can occurrandomly or at precisely defined sites, such as through homologousrecombination. For example, a functional copy of a trefoil familymolecule can be inserted in replacement of an altered version in a cell,through homologous recombination. Further techniques include gene gun,liposome-mediated transfection, or cationic lipid-mediated transfection.Gene therapy can be accomplished by direct gene injection, or byadministering ex vivo prepared genetically modified cells expressing afunctional polypeptide.

Delivery of nucleic acids into viable cells can be effected ex vivo, insitu, or in vivo by use of vectors, and more particularly viral vectors(e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus),or ex vivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). Non-limiting techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,and the calcium phosphate precipitation method (see, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)).Introduction of a nucleic acid or a gene encoding a polypeptide of theinvention can also be accomplished with extrachromosomal substrates(transient expression) or artificial chromosomes (stable expression).Cells may also be cultured ex vivo in the presence of therapeuticcompositions of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapyvectors. A number of viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol.73(Pt 6):1533-6), adenovirus (Berkner (1992) Curr Top Microbiol Immunol.158:39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia andKapikian (1992) J Virol. 66(7):4407-12; Quantin et al., (1992) Proc NatlAcad Sci USA. 89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55;Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9;Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56),vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22),adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol.158:97-129; Ohi et al., (1990) Gene. 89(2):279-82), herpesvirusesincluding HSV and EBV (Margolskee (1992) Curr Top Microbiol Immunol.158:67-95; Johnson et al., (1992) Brain Res Mol Brain Res.12(1-3):95-102; Fink et al., (1992) Hum Gene Ther. 3(1):11-9;Breakefield and Geller (1987) Mol Neurobiol. 1(4):339-71; Freese et al.,(1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian(Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54; Petropouloset al., (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) MolCell Biol. 12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6;Sorge et al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore(1985) J Virol. 54(2):401-7; Miller et al., (1988) J Virol.62(11):4337-45), and human origin (Shimada et al., (1991) J Clin Invest.88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page etal., (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) JVirol. 66(5):2731-9).

Non-limiting examples of in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors (see U.S. Pat.No. 5,252,479, which is incorporated by reference in its entirety) andviral coat protein-liposome mediated transfection (Dzau et al., Trendsin Biotechnology 11:205-210 (1993), incorporated entirely by reference).For example, naked DNA vaccines are generally known in the art; seeBrower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporatedby reference in its entirety. Gene therapy vectors can be delivered to asubject by, for example, intravenous injection, local administration(see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see,e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al.,Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469,6,017,524, 6,143,290, 6,410,010 6,511,847; 8,398,968; and 8,404,653which are all hereby incorporated by reference in their entireties. Foran example of gene therapy treatment in humans see Porter et al., NEJM2011 365:725-733 and Kalos et al. Sci. Transl. Med. 2011. 201 3(95):95.For additional reviews of gene therapy technology, see Friedmann,Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990);Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci.2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August;7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87;Jensen et al., Ann Med. 2007; 39(2):108-15; Herweijer et al., Gene Ther.2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31;10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005;99:193-260, all of which are hereby incorporated by reference in theirentireties.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application is understood by theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

Protein Delivery Methods.

Protein replacement therapy can increase the amount of protein byexogenously introducing wild-type or biologically functional protein byway of infusion. A replacement polypeptide can be synthesized accordingto known chemical techniques or may be produced and purified via knownmolecular biological techniques. Protein replacement therapy has beendeveloped for various disorders. For example, a wild-type protein can bepurified from a recombinant cellular expression system (e.g., mammaliancells or insect cells-see U.S. Pat. No. 5,580,757 to Desnick et al.;U.S. Pat. Nos. 6,395,884 and 6,458,574 to Selden et al.; U.S. Pat. No.6,461,609 to Calhoun et al.; U.S. Pat. No. 6,210,666 to Miyamura et al.;U.S. Pat. No. 6,083,725 to Selden et al.; U.S. Pat. No. 6,451,600 toRasmussen et al.; U.S. Pat. No. 5,236,838 to Rasmussen et al. and U.S.Pat. No. 5,879,680 to Ginns et al.), human placenta, or animal milk (seeU.S. Pat. No. 6,188,045 to Reuser et al.), or other sources known in theart. After the infusion, the exogenous protein can be taken up bytissues through non-specific or receptor-mediated mechanism.

A trefoil family molecule can also be delivered in a controlled releasesystem. For example, the trefoil family molecule can be administeredusing intravenous infusion, an implantable osmotic pump, a transdermalpatch, liposomes, or other modes of administration. In one embodiment, apump can be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al.(1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574).In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al. (1985) Science 228:190; During et al. (1989) Ann.Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105). In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlledrelease systems are discussed in the review by Langer (Science (1990)249:1527-1533).

Pharmaceutical Compositions and Methods of Administration

In some embodiments, a trefoil family molecule can be supplied in theform of a pharmaceutical composition, comprising an isotonic excipientprepared under sufficiently sterile conditions for human administration.Choice of the excipient and any accompanying elements of the compositioncomprising a trefoil family molecule will be adapted in accordance withthe route and device used for administration. In some embodiments, acomposition comprising a trefoil family molecule can also comprise, orbe accompanied with, one or more other ingredients that facilitate thedelivery or functional mobilization of the trefoil family molecule.

These methods described herein are by no means all-inclusive, andfurther methods to suit the specific application is understood by theordinary skilled artisan. Moreover, the effective amount of thecompositions can be further approximated through analogy to compoundsknown to exert the desired effect.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

A trefoil family molecule (such as, e.g., TFF1, TFF2, and TFF3) can beadministered to the subject one time (e.g., as a single injection ordeposition). Alternatively, a trefoil family molecule can beadministered once or twice daily to a subject in need thereof for aperiod of from about 2 to about 28 days, or from about 7 to about 10days, or from about 7 to about 15 days. It can also be administered onceor twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 times per year, or a combination thereof. Furthermore, atrefoil family molecule can be co-administrated with anothertherapeutic.

In one embodiment, a trefoil family molecule can be co-administratedwith a chemotherapy drug. Some non-limiting examples of conventionalchemotherapy drugs include: aminoglutethimide, amsacrine, asparaginase,bcg, anastrozole, bleomycin, buserelin, bicalutamide, busulfan,capecitabine, carboplatin, camptothecin, chlorambucil, cisplatin,carmustine, cladribine, colchicine, cyclophosphamide, cytarabine,dacarbazine, cyproterone, clodronate, daunorubicin, diethylstilbestrol,docetaxel, dactinomycin, doxorubicin, dienestrol, etoposide, exemestane,filgrastim, fluorouracil, fludarabine, fludrocortisone, epirubicin,estradiol, gemcitabine, genistein, estramustine, fluoxymesterone,flutamide, goserelin, leuprolide, hydroxyurea, idarubicin, levamisole,imatinib, lomustine, ifosfamide, megestrol, melphalan, interferon,irinotecan, letrozole, leucovorin, ironotecan, mitoxantrone, nilutamide,medroxyprogesterone, mechlorethamine, mercaptopurine, mitotane,nocodazole, octreotide, methotrexate, mitomycin, paclitaxel,oxaliplatin, temozolomide, pentostatin, plicamycin, suramin, tamoxifen,porfimer, mesna, pamidronate, streptozocin, teniposide, procarbazine,titanocene dichloride, raltitrexed, rituximab, testosterone,thioguanine, vincristine, vindesine, thiotepa, topotecan, tretinoin,vinblastine, trastuzumab, and vinorelbine.

In one embodiment, the chemotherapy drug is an alkylating agent, anitrosourea, an anti-metabolite, a topoisomerase inhibitor, a mitoticinhibitor, an anthracycline, a corticosteroid hormone, a sex hormone, ora targeted anti-tumor compound.

A targeted anti-tumor compound is a drug designed to attack cancer cellsmore specifically than standard chemotherapy drugs can. Most of thesecompounds attack cells that harbor mutations of certain genes, or cellsthat overexpress copies of these genes. In one embodiment, theanti-tumor compound can be imatinib (Gleevec), gefitinib (Iressa),erlotinib (Tarceva), rituximab (Rituxan), or bevacizumab (Avastin).

An alkylating agent works directly on DNA to prevent the cancer cellfrom propagating. These agents are not specific to any particular phaseof the cell cycle. In one embodiment, alkylating agents can be selectedfrom busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide,ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard),melphalan, and temozolomide.

An antimetabolite makes up the class of drugs that interfere with DNAand RNA synthesis. These agents work during the S phase of the cellcycle and are commonly used to treat leukemia, tumors of the breast,ovary, and the gastrointestinal tract, as well as other cancers. In oneembodiment, an antimetabolite can be 5-fluorouracil, capecitabine,6-mercaptopurine, methotrexate, gemcitabine, cytarabine (ara-C),fludarabine, or pemetrexed.

Topoisomerase inhibitors are drugs that interfere with the topoisomeraseenzymes that are important in DNA replication. Some examples oftopoisomerase I inhibitors include topotecan and irinotecan while somerepresentative examples of topoisomerase II inhibitors include etoposide(VP-16) and teniposide.

Anthracyclines are chemotherapy drugs that also interfere with enzymesinvolved in DNA replication. These agents work in all phases of the cellcycle and thus, are widely used as a treatment for a variety of cancers.In one embodiment, an anthracycline used with respect to the inventioncan be daunorubicin, doxorubicin (Adriamycin), epirubicin, idarubicin,or mitoxantrone.

In one embodiment, a trefoil family molecule can be co-administratedwith an anti-inflammatory drug. Some non-limiting examples ofanti-inflammatory drugs include: anti-inflammatory steroids(corticosteroids) (e.g. prednisone), aminosalicylates (e.g.,mesalazine), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g.aspirin, ibuprofen, naproxen) and immune selective anti-inflammatoryderivatives (ImSAIDs). An anti-inflammatory drug also includesantibodies or molecules that target cytokines and chemokines including,but not limited to, anti-TNFα antibodies (e.g. infliximab (Remicade),adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi),etanercept (Enbrel)), anti-IL12 antibodies, anti-IL2 antibodies(basiliximab (Simulect), daclizumab (Zenapax), azathioprine (Imuran®,Azasan®), 6-mercaptopurine (6-MP, Purinethol®), cyclosporine A(Sandimmune®, Neoral®), tacrolimus (Prograf®), and anti-GM-CSFantibodies.

In one embodiment, a trefoil family molecule can be co-administratedwith radiation therapy. Some non-limiting examples of conventionalradiation therapy include: external beam radiation therapy, sealedsource radiation therapy, unsealed source radiation therapy, particletherapy, and radioisotope therapy.

In one embodiment, a trefoil family molecule can be co-administratedwith a cancer immunotherapy. Cancer immunotherapy comprises using theimmune system of the subject to treat a cancer. For example, the immunesystem of a subject can be stimulated to recognize and eliminate cancercells. Some non-limiting examples of cancer immunotherapy include:cancer vaccines, therapeutic antibodies, such as monoclonal antibodytherapy (e.g., Bevacizumab, Cetuximab, and Panitumumab), cell basedimmunotherapy, and adoptive cell based immunotherapy.

A trefoil family molecule may also be used in combination with surgicalor other interventional treatment regimens used for the treatmentdisease of the digestive system.

A trefoil family molecule can be administered to a subject by any meanssuitable for delivering the protein, nucleic acid or compound to cellsof the subject. For example, it can be administered by methods suitableto transfect cells. Transfection methods for eukaryotic cells are wellknown in the art, and include direct injection of the nucleic acid intothe nucleus or pronucleus of a cell; electroporation; liposome transferor transfer mediated by lipophilic materials; receptor mediated nucleicacid delivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

The compositions of this invention can be formulated and administered toreduce the symptoms associated with a disease of the digestive system byany means that produce contact of the active ingredient with the agent'ssite of action in the body of a human or non-human subject. For example,the compositions of this invention can be formulated and administered toreduce the symptoms associated with an inflammatory disease of thedigestive system, a digestive system cancer, or a dysplasia of thedigestive system, or cause a decrease in cell proliferation, or adecrease in tumor growth. They can be administered by any conventionalmeans available for use in conjunction with pharmaceuticals, either asindividual therapeutic active ingredients or in a combination oftherapeutic active ingredients. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

Pharmaceutical compositions for use in accordance with the invention canbe formulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. The therapeutic compositions of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally can be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. (20^(th) ed., 2000), theentire disclosure of which is herein incorporated by reference. Forsystemic administration, an injection is useful, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the therapeutic compositions of the invention can beformulated in liquid solutions, for example in physiologicallycompatible buffers, such as PBS, Hank's solution, or Ringer's solution.In addition, the therapeutic compositions can be formulated in solidform and redissolved or suspended immediately prior to use. Lyophilizedforms are also included. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.These pharmaceutical formulations include formulations for human andveterinary use.

Any of the therapeutic applications described herein can be applied toany subject in need of such therapy, including, for example, a mammalsuch as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, asheep, a goat, or a human.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecomposition must be sterile and fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,a pharmaceutically acceptable polyol like glycerol, propylene glycol,liquid polyetheylene glycol, and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid, andthimerosal. In many cases, it can be useful to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating thetrefoil family molecule in the required amount in an appropriate solventwith one or a combination of ingredients enumerated herein, as required,followed by filtered sterilization. Dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions include an inert diluent or an edible carrier. Theycan be enclosed in gelatin capsules or compressed into tablets. For thepurpose of oral therapeutic administration, the active compound can beincorporated with excipients and used in the form of tablets, troches,or capsules. Oral compositions can also be prepared using a fluidcarrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as known in the art

A composition of the invention can be administered to a subject in needthereof. Subjects in need thereof can include but are not limited to,for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, amonkey, a pig, a sheep, a goat, or a human.

A composition of the invention can also be formulated as a sustainedand/or timed release formulation. Such sustained and/or timed releaseformulations can be made by sustained release means or delivery devicesthat are well known to those of ordinary skill in the art, such as thosedescribed in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123;4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of whichare each incorporated herein by reference. The pharmaceuticalcompositions of the invention (e.g., that have a therapeutic effect) canbe used to provide slow or sustained release of one or more of theactive ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or thelike, or a combination thereof to provide the desired release profile invarying proportions. Suitable sustained release formulations known tothose of ordinary skill in the art, including those described herein,can be readily selected for use with the pharmaceutical compositions ofthe invention. Single unit dosage forms suitable for oraladministration, such as, but not limited to, tablets, capsules,gel-caps, caplets, or powders, that are adapted for sustained releaseare encompassed by the invention.

In the methods described herein, a trefoil family molecule, can beadministered to the subject either as RNA, in conjunction with adelivery reagent, or as a nucleic acid (e.g., a recombinant plasmid orviral vector) comprising sequences which express the gene product.Suitable delivery reagents for administration of the a trefoil familymolecule, include the Minis Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; or polycations (e.g., polylysine), orliposomes.

The dosage administered can be a therapeutically effective amount of thecomposition sufficient to result in treatment of an inflammatory diseaseof the digestive system, treatment of an of a digestive system cancer, adecrease in cell proliferation, a decrease in tumor growth, or treatmentof dysplasia of the digestive system, and can vary depending upon knownfactors such as the pharmacodynamic characteristics of the activeingredient and its mode and route of administration; time ofadministration of active ingredient; age, sex, health and weight of therecipient; nature and extent of symptoms; kind of concurrent treatment,frequency of treatment and the effect desired; and rate of excretion.

In some embodiments, the effective amount of the administered trefoilfamily molecule is at least about 0.01 μg/kg body weight, at least about0.025 μg/kg body weight, at least about 0.05 μg/kg body weight, at leastabout 0.075 μg/kg body weight, at least about 0.1 μg/kg body weight, atleast about 0.25 μg/kg body weight, at least about 0.5 μg/kg bodyweight, at least about 0.75 μg/kg body weight, at least about 1 μg/kgbody weight, at least about 5 μg/kg body weight, at least about 10 μg/kgbody weight, at least about 25 μg/kg body weight, at least about 50μg/kg body weight, at least about 75 μg/kg body weight, at least about100 μg/kg body weight, at least about 150 μg/kg body weight, at leastabout 200 μg/kg body weight, at least about 250 μg/kg body weight, atleast about 300 μg/kg body weight, at least about 350 μg/kg body weight,at least about 400 μg/kg body weight, at least about 450 μg/kg bodyweight, at least about 500 μg/kg body weight, at least about 550 μg/kgbody weight, at least about 600 μg/kg body weight, at least about 650μg/kg body weight, at least about 700 μg/kg body weight, at least about750 μg/kg body weight, at least about 800 μg/kg body weight, at leastabout 850 μg/kg body weight, at least about 900 μg/kg body weight, atleast about 950 μg/kg body weight, at least about 1000 μg/kg bodyweight, at least about 1500 μg/kg body weight, at least about 2000 μg/kgbody weight, at least about 2500 μg/kg body weight, at least about 3000μg/kg body weight, at least about 3500 μg/kg body weight, at least about4000 μg/kg body weight, at least about 4500 μg/kg body weight, at leastabout 5000 μg/kg body weight, at least about 5500 μg/kg body weight, atleast about 6000 μg/kg body weight, at least about 6500 μg/kg bodyweight, at least about 7000 μg/kg body weight, at least about 7500 μg/kgbody weight, at least about 8000 μg/kg body weight, at least about 8500μg/kg body weight, at least about 9000 μg/kg body weight, at least about9500 μg/kg body weight, or at least about 10000 μg/kg body weight.

In one embodiment, a trefoil family molecule is administered at leastonce daily. In another embodiment, a trefoil family molecule isadministered at least twice daily. In some embodiments, a trefoil familymolecule is administered for at least 1 week, for at least 2 weeks, forat least 3 weeks, for at least 4 weeks, for at least 5 weeks, for atleast 6 weeks, for at least 8 weeks, for at least 10 weeks, for at least12 weeks, for at least 18 weeks, for at least 24 weeks, for at least 36weeks, for at least 48 weeks, or for at least 60 weeks. In furtherembodiments, a trefoil family molecule is administered in combinationwith a second therapeutic agent.

Toxicity and therapeutic efficacy of therapeutic compositions of thepresent invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Therapeutic agentsthat exhibit large therapeutic indices are useful. Therapeuticcompositions that exhibit some toxic side effects can be used.

Experimental animals can be used as models for human disease. Forexample, mice can be used as a mammalian model system. The physiologicalsystems that mammals possess can be found in mice, and in humans, forexample. Certain diseases can be induced in mice by manipulating theirenvironment, genome, or a combination of both. For example, the AOM/DSSmouse model is a model for human colon cancer. In another example, theDSS mouse model is a model for human colitis. Other mouse models ofcarcinogenesis include the two-stage DMBA/TPA model of skin cancer, theDEN/CCL4 model of liver cancer, and the H. felis/MNU model of gastriccancer. In addition, there are numerous genetically engineered models ofcancer, such as the KPC model of pancreatic cancer.

Administration of a trefoil family molecule is not restricted to asingle route, but may encompass administration by multiple routes.Multiple administrations may be sequential or concurrent. Other modes ofapplication by multiple routes will be apparent to one of skill in theart.

Methods of Detection

Embodiments of the invention provide for detecting expression of atrefoil family molecule (such as, e.g., TFF1, TFF2, TFF3). In oneembodiment, a gene alteration can result in increased or reduced proteinexpression and/or activity. The alteration can be determined at thelevel of the DNA, RNA, or polypeptide.

In some embodiments, the detecting comprises detecting in a biologicalsample whether there is a reduction in an mRNA encoding a trefoil familyprotein, or a reduction in a trefoil family protein, or a combinationthereof. In further embodiments, the detecting comprises detecting in abiological sample whether there is a reduction in an mRNA encoding atrefoil family protein, or a reduction in a trefoil family protein, or acombination thereof. The presence of such an alteration is indicative ofthe presence or predisposition to a digestive system cancer (e.g., coloncancer) or an inflammatory disease of the digestive system.

Methods for detecting and quantifying trefoil family molecules, (suchas, e.g., TFF1, TFF2, TFF3 proteins and polynucleotides) in biologicalsamples are known the art. For example, protocols for detecting andmeasuring the expression of a polypeptide encoded by a trefoil familymolecule, such as TFF1, TFF2, TFF3, using either polyclonal ormonoclonal antibodies specific for the polypeptide are well established.Non-limiting examples include Western blot, enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA), and fluorescence activated cellsorting (FACS).

In one embodiment, a biological sample comprises, a blood sample, serum,cells (including whole cells, cell fractions, cell extracts, andcultured cells or cell lines), tissues (including tissues obtained bybiopsy), body fluids (e.g., urine, sputum, amniotic fluid, synovialfluid), or from media (from cultured cells or cell lines). The methodsof detecting or quantifying trefoil family molecules (such as, e.g.,TFF1, TFF2, TFF3) include, but are not limited to, amplification-basedassays with (signal amplification) hybridization based assays andcombination amplification-hybridization assays. For detecting andquantifying trefoil family molecules (such as, e.g., TFF1, TFF2, TFF3),an exemplary method is an immunoassay that utilizes an antibody or otherbinding agents that specifically bind to a trefoil family protein (suchas, e.g., TFF1, TFF2, or TFF3) or epitope of such, for example, Westernblot or ELISA assays.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1 Generation of TFF2 Transgenic Mice

A knock out the trefoil factor family 2 (TFF2) gene in mice showed thatloss of TFF2 resulted in increased inflammation in response to DSScolitis or Helicobacter gastritis, suggesting that TFF2 dampenedinflammatory responses. In addition, other results showed that TFF2 wasspecifically silenced in many cancers, suggesting it was a tumorsuppressor gene. Most recently, it was shown that TFF2 is normallyexpressed by a subset of T cells, and that such TFF2 expression acts toregulate myeloid progenitors. In response to the induction of cancer,myeloid progenitors proliferate and are markedly amplified, resulting inincrease myeloid derived suppressor cells (MDSC) that promote cancergrowth. Thus, in TFF2 knockout mice these cells are much increased. ATFF2 overexpressing mouse was generated, where TFF2 was highly expressedin all T cells, and found that in response to carcinogens, myeloidproliferation was suppressed and the mice did not develop cancer.Accumulating evidence has indicated that blocking myeloid cell expansion(e.g. using an antibody to GM-CSF) can inhibit cancer initiation andprogression.

TFF2 can be used, delivered as a recombinant peptide or using a viralvector or modified peptide (for increased stability) to treat advancedcancer (or dysplasia) by specifically suppressing myeloid proliferationwith this approach. More potent means of delivery are being developed.

TFF2 can be a new and useful cancer therapy that works by targeting thetumor microenvironment, specifically the myeloid cells (e.g. MDSC, tumorassociated macrophages, neutrophils) that support cancer. In addition,it can be a form of replacement of a tumor suppressor gene product thatis normally downregulated in many cancers.

TFF2 differs from other myeloid therapies, such as anti-CSF oranti-GM-CSF, in that it would be a natural peptide and not a monoclonalantibody. While most useful potentially in treatment of advanced cancer,it can also be used in cancer prevention therapy in high riskindividuals.

Example 2 TFF2 is a Novel Tumor Suppressor that Inhibits Expansion ofGr1+CD11b+ Myeloid Derived Suppressor Cells (MDSC) and Blocks ColonCarcinogenesis

Trefoil factor 2 (TFF2) is a small secreted protein that is expressed ingastrointestinal mucosa where it functions to protect and repair mucosa.It is, however, also expressed at low levels in splenic immune cellswhere its role has been unclear. TFF2 is epigenetically silenced ingastric cancer and thus has been postulated to protect against cancerdevelopment through multiple mechanisms. Since TFF2 is normallyexpressed in splenic T-cells the specific contribution of T-cell relatedTFF2 production in the modulation of tumorigenesis was investigated. Itwas discovered that TFF2 is a critical modulator of the aberrantinflammatory response that promotes carcinogenesis.

Methods:

Transgenic (TG) mice that overexpress TFF2 under the control of thehuman CD2 promoter (specific to T-cells) were created. These TG micewere compared to TFF2−/− (knockout) and wild-type (WT) mice ininflammation and inflammatory carcinogenesis models (including the DSScolitis model and the AOM/DSS colon cancer model). The contribution ofT-cell TFF2 production on tumor development and the associated immuneresponse was examined in vivo and mechanisms analyzed in vitro.

Results:

DSS colitis caused a marked early (day 1-3) upregulation of TFF2production in the spleen, absent in TFF2−/− mice. Compared to the WTmice, the null mice displayed a dramatically amplified inflammatoryresponse to DSS with increased splenic cell proliferation and associatedincreases in MDSCs (Gr1+CD11b+) detected in both the spleen and bonemarrow. In contrast, the proliferation and expansion of Gr1+CD11b+ cellsseen with DSS was markedly suppressed in the TFF2 overexpressing TGmice. This was consistent with an immune modulatory role of T-cell TFF2.Interestingly, this aberrant inflammatory response to DSS seen in theTFF2 null mice, translated into an ordered difference in ultimate tumordevelopment in the AOM/DSS model with TFF2−/− mice developing morecolonic tumors then WT mice, which in turn developed more than the TGmice. The TG mice showed almost complete suppression of colonictumorigenesis (P<0.05). To identify the cellular target of TFF2, aeukaryotic TFF2 expression construct was generated using the pMIG vectorto express mouse TFF2 (mTFF2) in CHO-KI cells. Recombinant mTFF2 wasproperly folded in this system and subsequently purified. It was foundthat mTFF2 suppressed, in a dose-dependent manner, the proliferation ofGr1+CD11b+ cells (MDSCs) in vitro. Thus, potentially revealing themechanism through which TFF2 modulated the immune response and reducedinflammatory carcinogenesis.

Conclusion:

Overexpression of TFF2 markedly suppressed tumor growth by curtailingthe proliferation and expansion of myeloid progenitors that give rise toMDSCs. This novel mechanism for suppressing myeloid cells may haveimplications for cancer prevention and therapy.

Example 3 TFF2 Inhibits Expansion of Gr1+CD11b+Myeloid-DerivedSuppressor Cells and Blocks Colon Carcinogenesis

Trefoil factor 2 (TFF2) is a small secreted protein that is expressed ingastrointestinal mucosa where it functions to protect and repair mucosa,but it is also expressed at low levels in splenic immune cells where itsrole has been unclear. The Tff2 gene is epigenetically silenced ingastric cancers and thus has been postulated to protect against cancerdevelopment through multiple mechanisms.

The aims include:

-   -   Identify the cell target of TFF2 in suppressing tumor        development.    -   Determine the specific contribution of T cell-derived TFF2 in        the modulation of tumorigenesis.

Methods

Transgenic (TG) mice that overexpress TFF2 under the control of thehuman CD2 promoter, T cells-specific promoter were created. These TGmice were compared to Tff2-−/− and wild-type (WT) mice in inflammationand inflammatory carcinogenesis models (including the DSS—inducedcolitis model and the AOM/DSS colon cancer model). The contribution of Tcell derived TFF2 on tumor development and the associated immuneresponse was examined in vivo and mechanisms analyzed in vitro.

Results

DSS administration (colitis) caused a marked early (day 1-3)upregulation of TFF2 expression (production) in the spleen. Compared tothe WT mice, TFF2−/− mice displayed worse inflammatory response to DSSwith increased proportion of Gr1+CD11b+ cells (myeloid-derivedsuppressor cells [MDSCs]) in the spleen and bone marrow. In contrast,the proliferation and expansion of Gr1+CD11b+ cells seen with DSStreatment was markedly suppressed in the TFF2 overexpressing TG mice.Consistently TFF2−/− mice developed more colonic tumors than WT mice,which in turn developed more than the TG mice in AOM/DSS model. The TGmice showed almost complete suppression of colonic tumorigenesis(p<0.05) with normal proportion of MDSCs in spleen in contrast toTFF2−/− deficient mice which all develop tumor and display expansion ofIMC in spleen and bone marrow. To identify the cellular target of TFF2,a eukaryotic TFF2 expression construct was generated using the pMIGvector to express mouse TFF2 (mTFF2) in CHO-KI cells. Recombinant mTFF2was properly expressed in this system and subsequently purified. Wefound that recombinant mTFF2 suppressed, in a dose-dependent manner, theproliferation of Gr1+CD11b+ cells (MDSCs) in vitro, thus potentiallyrevealing the mechanism through which TFF2 modulates the immune responseand reduces inflammatory carcinogenesis.

Conclusion

Overexpression of TFF2 markedly suppressed tumor growth by curtailingthe proliferation and expansion of myeloid progenitors that give rise toMDSCs. This novel mechanism for suppressing myeloid cells may haveimplications for cancer prevention and therapy.

Example 4 TFF2 Inhibits Expansion of Gr1+CD11b+ Myeloid-DerivedSuppressor Cells and Blocks Colon Carcinogenesis

Trefoil factor 2 (TFF2) is a small secreted protein that is expressed ingastrointestinal mucosa where it functions to protect and repair mucosa.It is also expressed at low levels in splenic immune cells where itsrole has been unclear. The Tff2 gene is epigenetically silenced ingastric cancers and thus has been postulated to protect against cancerdevelopment through multiple mechanisms.

Methods

The aims include:

-   -   Determine the specific contribution of T cell-derived TFF2 in        carcinogenesis.    -   Identify the cells targeted by TFF2 to reduce tumor development.

Generation of Transgenic Mice

Transgenic (TG) mice that overexpress TFF2 under the control of thehuman CD2 promoter (a T cell-specific promoter, FIG. 2A) were created.These TG mice were compared to TFF2−/− and wild-type (WT) mice ininflammation and inflammatory carcinogenesis models (including theDSS—induced colitis model and the AOM/DSS colon cancer model). Thecontribution of T cell derived TFF2 on tumor development and theassociated immune response was examined in vivo and mechanisms analyzedin vitro.

Generation of Mice Chimaeras

WT mice were lethally irradiated and transplanted with bone marrow fromWT, TFF2−/− and TG mice. The chimaeras were given 5% DSS water for 5days and tap water for the remaining days. They were sacrificed on day19 and differences in clinical disease parameters were measured.

Results

TFF2 is Upregulated Upon Splenic T Cells Activation

TFF2 is expressed in the stomach and spleen of WT mice (FIG. 1B). RobustTFF2 mRNA expression was detected in resting splenic T cells from WTmice (FIG. 1C). There was minimal expression in B cells at baseline.When murine splenocytes were stimulated with concavalin A (Con A), a Tcell-specific stimulant, and lipopolysaccharides (LPS), a Bcell-activator, a 40- and 2.5-fold increase was observed in TFF2 mRNAexpression respectively over unstimulated splenocytes (FIG. 1D). Anupregulation of TFF2 expression in WT spleen was also detected afteradministration of 3% DSS water (FIG. 1F).

TFF2−/− Mice have More Severe DSS-Induced Inflammation

TFF2 deficient mice had more severe inflammation and higher mortalityrate than TG and WT mice after DSS administration (FIG. 3A). TFF2−/−mice had greater reactive splenomegaly compared to TG and WT upon 2.5%DSS regimen (FIG. 3E).

Transgenic Mice Overexpressing TFF2 in T Cells (TG Mice) Show AttenuatedDSS Colitis

Unlike TFF2−/− mice, TG mice showed attenuated DSS colitis. Tff2−/−showed an upregulation of inflammatory cytokines IL-1b at day 19 (FIG.4A) and myeloperoxidase (MPO) activity at acute phase of colitis on day6 after DSS administration (FIG. 4E) suggesting the role of TFF2 inmodulating inflammation.

WT Mice Reconstituted with Bone Marrow from TFF2−/− Showed More AcuteInflammatory Response

Following bone marrow (BM) transplantation and DSS, WT recipients ofTFF2−/− donor BM were most affected. They lost more weight (FIG. 5A),had shorter colons (FIG. 5B), more severe splenomegaly (FIG. 5D) andgreater increase of IL-1β in colon (FIG. 5E) at day 19 compared torecipients of TG or WT BM. Highlighting the importance of hematopoieticTFF2 production.

DSS Treatment Increased Gr1+CD11b+(Myeloid-Derived Suppressor Cells,MDSCs) Cells in the Spleen of TFF2−/− and WT but not in the Spleen of TGMice

In comparison to WT and TG groups, TFF2−/− mice had more significantsplenic myeloid proliferation following DSS treatment ((examined by Ki67(brown, proliferation) and Gr1 (red, myeloid marker) coimmunostaining)(FIG. 7A). Coimmunostaining was localized to the red pulp zone, wheremyeloid cells reside (FIG. 7B). FACS analysis showed TFF2expression-dependent expansion of CD11b+Gr1+ cells in the spleen andbone marrow of the mice (FIGS. 7C, 7D) after DSS-treatment. Thus,splenomegaly in DSS-treated TFF2−/− mice resulted from the expansion ofimmature myeloid cells due to extramedullary hematopoiesis.

TFF2 Inhibits Proliferation of Gr1+CD11b+ Cells

Using BrdU uptake as another measure of proliferation, it was found thatfollowing DSS TFF2−/− mice showed greatest, WT mice intermediate and TGmice least proliferation of Gr1+CD11b+ cells (D+19, FIG. 8A). Gr1+CD11b+cells cultured with increasing concentrations of rTFF2 (murine) alsoshowed a dose-dependent decrease in viability (FIG. 8C).

Expression of TFF2 by Splenic T Cells Suppressed the Development ofColon and Rectal Tumors in AOM/DSS Model.

AOM/DSS treatment showed a similar gradient of Gr1+CD11b+ cell number inBM, spleen and blood relative to the TFF2 sufficiency of the host(TFF2−/− >WT>TG, FIG. 11D). TFF2−/− mice developed more colonic tumors(FIG. 11A, B) and increased colonic inflammation (FIG. 11C) than WT andTG mice following AOM/DSS model of colonic tumorigenesis.

Conclusion

TFF2 suppresses tumorigenesis by inhibiting the expansion of MDSCs. Thisnovel mechanism has implications for cancer prevention and therapy.

Example 5 TFF2 Secreted by Splenic T-Cells Dampens Inflammation andInhibits Carcinogenesis Through Suppression of Immature CD11b+Gr1+Myeloid Cells

Trefoil factor 2 (TFF2) is a small protease resistant peptide secretedby the stomach that plays a prominent role in mucosal protection. Here,evidence that TFF2 is also produced by lymphoid T-cells and suppressestumorigenesis associated with inflammation is provided. Transgenic mice(CD2-TFF2) overexpressing TFF2 in splenic T-cells are more resistant toDSS colitis, while TFF2-deficient mice show greater systemic and colonicinflammatory responses. Transplant of TFF2-deficient bone marrow intowild-type mice reproduces the DSS-injury susceptibility phenotype, whiletransplant of bone marrow from CD2-TFF2 transgenic mice reducesinflammatory responses. Following DSS treatment, TFF2-deficient miceaccumulate a greater number of Gr1+CD11b+ immature myeloid cells (IMC)in the spleen and bone marrow, while CD2-TFF2 transgenic mice showminimal increases in splenic IMC. The expansion of splenic IMC inTFF2-deficient mice was associated with higher number ofgranulocyte-macrophage precursors and an increase in proliferating Gr-1+myeloid cells, which was suppressed in CD2-TFF2 transgenic mice.Consistently, number of colony-forming units obtained from spleen ofTFF2−/−-deficient mice has been found significantly higher while in TGmice it was much less compare with wild type counterparts. An additionof recombinant TFF2 suppressed proliferation of Gr1+CD11b+ cells invitro experiments. Furthermore, all TFF2−/− deficient mice develop colontumors in model AOM/DSS and show higher tumor burden compared with wildtype mice and transgenic mice. Only 30% of transgenic mice developtumors with a very low tumor burden. The number of colony-forming unitsin the spleen of TFF2 knockout mice was found to be much higher comparedwith wild-type while transgenic mice showed the lowest number ofcolony-forming units and granulocyte-macrophage cells as well. Takentogether, these data show that TFF2 restricts expansion of Gr1+CD11b+cells through inhibition of proliferation of myeloidprogenitors/precursors. This accounts for less tumorigenesis associatedwith inflammation.

Introduction

Tumor growth and progression can be accompanied by expansion ofmyeloid-derived suppressive cells (MDSCs) that are commonlycharacterized as a heterogeneous population expressing surface markersGr1 and CD11b in mice. These cells suppress host immune response throughinhibition of T-cells and natural killer cells function (1). At presentthere are several known tumor-derived factors that regulate the functionand biology of MDSC. Growth factors GM-CSF and G-CSF along with cytokineIL-6 greatly modulate suppressive functions of myeloid cells throughtranscription factor C/EBPβ (2-5). IL-1b, TGF-b (6,7), IL-10, vascularendothelial growth factor (8), and prostaglandin E2 are also identifiedas factors promoting expansion of Gr1+CD11b+ cells.

The trefoil factor family in mammals comprises a group of three secretedproteins that all contain a highly conserved triple loop structure (thetrefoil domain). In the gastrointestinal tract, trefoil factor family 1(TFF1) is normally produced in the epithelium of gastric surface pits,while trefoil peptide 2 (TFF2) is most abundant in the stomach/duodenum,where, along with TFF1, it plays a role in the maintenance of mucuslayer integrity as well as in stimulation of mucosal restitution, inpart through a motogenic effect on epithelial cells (9-11). TFF3 ishighly expressed in the apical part of goblet cells in the intestine andcolon but not in normal gastric mucosa (9-11).

TFF2 and TFF3-deficiency does not result in obviously changed phenotype,however these mice have increased susceptibility to DSS treatmentcompared with wild type counterparts (12,13). In contrast, allTFF1-deficient mice developed adenoma and 30% of them showed carcinoma(14,15). From clinical research it is well known that TFF1 expression islost in 40-60% of human gastric tumors (16). In addition, inactivationof TFF1 by deletion, missence mutation or promoter methylation resultsto tumor incidence in mice (17).

In contrast TFF1 the role of TFF2 as a gastric tumor suppressive genewas not so clear. However, the loss of TFF2 during progression ofintestinal-type gastric cancer in human samples has been also reported(18,19). Like TFF1 the downregulation of TFF2 expression occurred likelydue to aberrant promoter methylation (20,21). Importantly TFF2-deficientmice progressed more quickly to dysplasia in the setting of H. pyloriinfection or when crossed to gp130F/F mice (21,22). Considering directlink between cancer and inflammation and the anti-inflammatory nature ofTFF2 there is a potential role of TFF2 as a gastric tumor suppressivegene however, there is still no direct experimental proof of theanti-tumor effect of TFF2.

The anti-inflammatory effect of TFF2 has been proven in numerous studieson experimental rodent models with induced colitis although themechanism is not clearly understood. In contrast to the rat where TFF2expression is observed in colon tissue, in mice neither TFF2 peptide normRNA is produced in the large intestine, even under inflammatoryconditions (23). Nevertheless, administration of recombinant TFF2ameliorates the severity of experimental colitis induced with bowelirritants such as dextran sodium sulfate (DSS), ethanol or indomethacinin rodents (24-28). Additionally, TFF2-deficient mice have more severeDSS-induced colitis (and delayed recovery) in comparison with wild typeanimals (13). Analysis of the distribution of endogenous trefoilpeptides along the various compartments of the gastrointestinal tractrevealed that TFF2 peptide is detectable in normal human luminalcontents from the distal and proximal part of the gut. This findingsuggests an effective transit and remarkable stability of gastric TFF2peptide along the whole gastrointestinal tract (29,30). Presumably,gastric TFF2 may exert its protective effect in mouse colitis model inpart due to a potentiation of mucin barrier function (31). Indeed,radiolabeled TFF2 injected intravenously in rats is specifically takenup by TFF2-producing cells and then transferred to the mucus where TFF2presumably mediates its protective function (10,32,33).

However, expression of trefoil factors is not restricted solely togastrointestinal epithelial cells. It has been shown TFF2 mRNAexpression at much lower levels in primary and secondary lymphaticorgans thymus and spleen, where their expression increased upon LPStreatment (13,34). Since then it has been suggested that trefoil factorsare intimately involved in the regulation of immune responses. Indeed,TFF2 exhibits chemotactic activity for human monocytes (34), inhibitsLPS-induced nitric oxide production by a monocyte cell line in vitro(35), inhibits myeloperoxidase activity in a model of DSS-inducedcolitis (25,28), and decreases leukocyte recruitment by reducing theexpression of vascular adhesion component-1 (VACM-1) (24). Furthermore,TFF2 deficiency results in the upregulation of expression of severalgenes that have been implicated in immune regulation, including MHCI,MHCII, cryptdin family members, etc. (36). In addition, splenic T cellsfrom TFF2-deficient mice were found to be hyper-responsive to IL-1βstimulation, suggesting a specific role in negative regulation of IL-1βreceptor—mediated signaling (13). Finally, it was recently demonstratedthat TFF2 was able to dampen SDF-1 induced signaling in vitro and invivo studies through CXCR4 receptor (37,38). Indeed, in transgenic miceoverexpression of SDF-1 in gastric mucosa increased gastric epithelialproliferation and hyperplasia, however TFF2-deficient mice crossed withtransgenic SDF-1 mice developed markedly more severe inflammation,hyperplasia and metaplasia conforming that TFF2 as a partial antagonistof SDF-1 in vivo. Very recently it has been shown that TFF2 throughCXCR4 receptor induces IL-33 release from lung epithelial, dendriticcells and macrophages resulting in the development of type 2 immuneresponse in asthma (39).

Nevertheless, data on effect of trefoil factors on function of primaryimmune cells are still limited, and most of them are derived primarilyfrom in vitro experiments that do not reproduce the inflammatorymicroenvironment in vivo (13,40). Moreover, it is possible that trefoilfactors modulate function of target cells indirectly by affecting othercell populations or through the interaction with partner(s). Included inthis example, the function of secreted TFF2 in the immune compartmentthrough the generation of a transgenic mouse bearing an expressioncassette consisting of mTFF2 open reading frame inserted under thecontrol of human CD2 gene promoter that targets T-cell specifictransgene expression (41) is explored. CD2-TFF2 transgenic mice andTFF2−/− deficient mice were used as experimental models to investigatethe role of TFF2 in colonic inflammation and colitis-associatedcancerogenesis (tumorigenesis associated with inflammation). Analysis ofthe CD2-TFF2 transgenic mice, along with the TFF2−/− mice, points to acritical role of TFF2 in the generation of myeloid-derived suppressorcells (MDSC) during inflammatory and carcinogenic stimuli. Without beingbound by theory there is strong evidence that myeloid Gr+CD11b+ cellsare the mediators/targets of TFF2 anti-inflammatory function in vivo.

Materials and Methods

Mice

Wild type C57/BL6 mice (7-12 weeks) were purchased from JacksonLaboratories (Bar Harbor, Me.), TFF2-deficient mice (TFF2−/−) on C57/BL6background were described earlier, CD2-TFF2 mice (TG) were generated incurrent study. Mice were group housed under a controlled temperature(25° C.) and photoperiod (12:12-h light-dark cycle). To discriminate therole of TFF2 expressed by epithelial and immune cells bone marrowtransplantation were performed using TFF2−/− deficient, WT and CD2-TFF2transgenic mice as recipients or donors of bone marrow (hematopoietic)cells in various combinations.

Cloning mTFF2 into hCD2 Cassette, Generation of CD2-TFF2 Transgenic Miceand Screening for CD2-TFF2 Transgene.

Mouse gene TFF2 was cloned downstream into hCD2 promoter into EcoRI siteof expressing vector. For this purpose the site for EcoRI wasincorporated in primers for PCR amplification of mouse TFF2 sequenceusing respective mouse cDNA library (Open Biosystem).

The forward primer was ATTG AATTC GCC ACC ATG CGA CCT CGA GAT GCC (SEQID NO: 7) (Tm=60.6C). The reverse primer was AATTG AATTC TCA GTA GTG ACAATC TTC CACAGA C (SEQ ID NO: 8) (Tm=56.2C) Site for EcoRI is shown inbold. PCR amplification produced 406 bp fragment of intact mTFF2. Aftercloning the resulting construct was transfected into E. coli Stb12competent cells. Clones were verified for presence and properorientation of TFF2 gene by sequencing using forward primer located inCD2 and reverse primer located within TFF2 sequence (see below).

Transgenic mouse lines expressing the TFF2 protein were generated inTransgenic Core Facility of Columbia University as follows. The DNA ofpCD2-TFF2 was digested by SalI/NotI to remove vector sequence and thefragment with CD2 cassette was purified by gel electrophoresis. Thefragment was microinjected into pronuclei of fertilized mouse eggs andthe injected embryos were implanted into pseudopregnant outbred females.

The offspring were screened for transgene integration by PCR analysis oftail DNA using primers selected for promoter part of CD2 gene and TFF2gene. Forward primer was 5′-TAAGCTCTCGGGGTGTGGACTC-3′ (SEQ ID NO: 9),Reverse primer was: GAAGTGGGTGGAAACACCAAGG (SEQ ID NO: 10). The correctsize of amplified fragment was 472 bp. PCR amplification was performedfor 30 cycles using following conditions: denaturation for 20 sec at 94°C., annealing at 65° C. for 20 sec, and elongation at 72° C. for 40 sec,followed by a final 7 min extension at 72° C.

Four founder mice were produced. These mice were bred with C57BL/6 mice.The offspring of the founders were tested for presence of transgenicCD2-TFF2 mRNA transcript expression in spleen and thymus as follows.Total RNA was isolated from whole spleen and thymus, using a Trizolreagent (Invitrogen-Life Technologies, Inc.) and RNAeasy® Mini Kit. Fivemicrograms of total RNA were reverse transcribed to cDNA withSuperscript III reverse transcriptase (Invitrogen, Carlsbad USA). PCRamplification was performed by using primers specific for TFF2 sequenceand for CD2 promoter part. Forward primer: 5′-TCTCCAAAGAATTCGCCACCAT-3′(SEQ ID NO: 11), reverse primer: 5′-GGTTGGAAAAGCAGCAGTTTCG-3′ (SEQ IDNO: 12), the predicted fragment size 351 bp. PCR amplification wasperformed for 30 cycles using the following conditions: denaturation for20 sec at 94° C., annealing at 56° C. for 30 sec, and elongation at 72°C. for 30 sec, followed by a final 7 min at 72° C. Two transgenic lineswere maintained by back-crossing with C57BL/6 for at least 10 timesbefore using in experiments.

Isolation of Splenic T- and B-Cells

Splenocytic T- and B-cells were isolated from resting splenocytes bynegative selection using immunomagnetic separation kit (MyltenyiBiotech, Inc.). To confirm the validity of the separation procedure theRNA was extracted from T- and B-cell population and subjected to PCRanalysis of cell-specific markers Thy 1.2 (marker for T-cells) and CD19(marker for B-cells) in respective populations.

Semi-Quantitative RT-PCR Analysis

A semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) method was used to measure the relative abundance of TFF2 mRNAtranscripts in resting total splenocytes and splenic B- and T-cells. RNAwas isolated from whole spleen, and separated splenic B-cells andT-cells, using a Trizol reagent (Invitrogen-Life Technologies, Inc.) andRNeasy® Mini Kit (Qiagen). 5 μg of total RNA was reverse transcribed tocDNA with Superscript III reverse transcriptase (Invitrogen, CarlsbadUSA).

PCR amplification was performed using the AB Applied Biosystem device,and amplified PCR products were analyzed in 1.5% agarose gel. All primerpairs, PCR conditions, and predicted sizes of amplified products arelisted below. Thy 1.2 antigen forward primer:5′-GCTGGACTGCCGCCATGAGAA-3′ (SEQ ID NO: 13) Thy 1.2 antigen reverseprimer: 5′-TGCCGC CACACTTGACCAGC-3′ (SEQ ID NO: 14), fragment size 295bp. PCR amplification was performed for 30 cycles using the followingconditions: denaturation for 20 s at 94° C., annealing at 68° C. for 5s, and elongation at 72° C. for 5 s, followed by a final 7 min at 72° C.CD19 forward primer: 5′-TGCTCAGCGTTGGGCTGCTG-3′ (SEQ ID NO: 15). CD19reverse primer: 5′-TGGGACCCAAGCGAGGATGC-3′ (SEQ ID NO: 16), fragmentsize 390 bp. PCR amplification was performed for 30 cycles using thefollowing conditions: denaturation/annealing for 30 s at 94° C., andelongation at 68° C. for 25 s, followed by a final 7 min at 72° C.

Mouse β-actin sense oligonucleotide: 5′-ACCACACCTTCTACAATGAGCTGC-3′ (SEQID NO:17). Mouse β-actin anti-sense oligonucleotide:5′-CTTCTCTTTGATGTCACGCACG-3′ (SEQ ID NO: 18), fragment size 386 bp. PCRamplification was performed for 25 cycles using the followingconditions: denaturation for 20 s at 94° C., annealing at 55° C. for 30s, and elongation at 72° C. for 30 s, followed by a final 7 min at 72°C. RNA was purified using Trizol® Reagent (Invitrogen, Scotland, UK) andRNeasy® Mini Kit. Total RNA was reverse transcribed with Superscript IIIreverse transcriptase (Invitrogen, Carlsbad USA). PCR amplification wasperformed for 25 cycles using the following conditions: denaturation for20 s at 94° C., annealing at 55° C. for 30 s, and elongation at 72° C.for 30 s, followed by a final 7 min at 72° C. For calculation of foldaugmentation RNA amounts were normalized to β-actin mRNA.

Western Blot Analysis of TFF2 Protein Level

Thymus (spleen) were homogenized in the loading buffer withβ-mercaptoethanol and then boiled for 5 min. Proteins were resolved in18% tris-glycine (or 10-20% Tris-Tricine) SDS-polyacrylamide gel andelectrophoretically transferred for 1 hour at 100V onto 0.2 μm-pore-sizePVDF membrane (Immobilon-P^(sq), Millipore). The filter was blocked 5%non-fat milk in 0.05% Tween 20/PBS (PBS-T) for 1 hour and incubatednight with primary rabbit antibodies (1 μg/ml) raised to C-end of TFF2(Tu et al., 2007) at 4° C. overnight. After washing in PBS-T theincubation was performed with secondary antibodies conjugated withhorseradish peroxidase (GE Healthcare, dilution 1:20000) for 25 min atroom temperature and after final washing the membrane was developed withSuperSignal West Femto Maximum Sensitivity Substrate Kit (Pierce ECL).

Quantitative Real-Time PCR for Cytokines

Total RNA was isolated from tissues using Trizol reagent (GIBCO BRL)according to the manufacturer's guidelines. Total RNA was purified usingthe RNeasy kit (QIAGEN). After DNase I (Invitrogen) treatment, 2 μg oftotal RNA was used for reverse transcription reaction (AppliedBiosystems). The specific primers for target genes were selected indifferent exons to avoid/minimize amplification of genomic DNA by usingprogram Primer 3 (http://fokker.wi.mit.edu/primer3/input.htm).Quantitative real-time PCR was performed on an AB 7300 System (AppliedBiosystems, Warrington, U.K.) using SYBR GREEN PCR Master Mix (AppliedBiosystems). Amplification conditions were: 50° C. (2 min), 95° C. (15min), 45 cycles of 95° C. (15 s), and 55° C. (15 s). The expression ofeach mRNA was normalized to housekeeping gene GAPDH mRNA expression, andsubsequently expressed as the fold change relative to non-inflamedcontrols.

Induction of Colitis

In the experiments involving DSS-induced colitis, 10 to 12-weeks oldsex-matched KO, TG and WT mice were used unless specified otherwise. Asa control KO, TG and WT mice received only ordinary water without DSS.For induction of chronic colitis, KO, TG and WT mice were given 2.5% DSS(m/w 36 000-50 000; MP Biomedicals, Solon, Ohio, USA) dissolved indrinking water provided ad libitum for 5 days, followed by provision ofplain water for 19 days. Mice were daily weighed, and blood in the stoolwas analyzed at days 3, 5, 7, 9, 11, 13 and 15 using Hemoccult strips(4-5 mice per each group). For stool consistency, a score of 0 pointswas assigned for well-formed pellets, 2 points for pasty and semiformedstools that did not adhere to the anus, and 4 points for liquid stoolsthat did adhere to the anus. For bleeding, a score of 0 points wasassigned for no blood, 2 points for positive hemoccult, and 4 points forgross bleeding. These scores were added together and divided by three,resulting in a total clinical score ranging from 0 (healthy) to 4(maximal activity of colitis).

For acute colitis induction mice received 2.5% DSS for 5 days andsacrificed at 6 day. In another set of experiments mice received 4% DSSfor 7 days followed by plain water for 12 days and were sacrificed at 19day.

Bone Marrow Transplantation

Bone marrow harvested from the femur and tibia was depleted oferythrocytes by using red blood cell lysis buffer (Sigma, R7757)according to manufacturer's instructions. Mice were lethally irradiatedwith 9Gy and after 3 h were reconstituted with bone marrow cells(3.5×10⁶) by using tail injection. For bone marrow chimaeras, wherewild-type and SPKO mice were reconstituted with transgenic and wild typebone marrow respectively the presence of mRNA and CD2-TFF2 transcriptswas analyzed by qRT-PCR to determine a degree of chimerism.Irradiatiated control mice which did not received transferred bonemarrow cells, served as an irradiation control in each experiment.Transplanted mice were allowed to rest 7-8 weeks for full engraftmentbefore DSS water was administrated for 5 days followed plain water. Micewere sacrificed on day 6 and 19 after starting DSS administration andanalyzed for progression of colitis (macroscopical and histologicalcolon examination, MPO activity, cytokines level, spleen mass, analysisof splenic cell population)

Assessment of Colitis

Progression of colitis was evaluated by disease activity index (DAI)derived by scoring body weight change (0-4), stool consistency (0-4),and by the presence or absence of fecal blood (0-3) (Xu Y, Hunt N H, BaoS, 2008). These scores were added together and divided by three,resulting in a total clinical score ranging from 0 (healthy) to 4(maximal activity of colitis). Mice were daily weighed and change inbody weight was calculated by the percentage change (gain/loss) from theinitial weight (0, less than 5% change; 1, 5-10%; 2, 10-20%; 4, morethan 20%). For stool consistency, a score of 0 points was assigned forwell-formed pellets, 2 points for pasty and semi formed stools that didnot adhere to the anus, and 4 points for liquid stools that did adhereto the anus (Blood in the stool was analyzed both visually and byHemoccult strip as described previously (Kourt-Jones et al., 2007).Scoring for blood in stool was as follows: 0, none; 1, trace usingHemoccult strips; 2, strong positive using Hemoccult strips; 3, grosshemorrhage.

Colonic inflammation was also evaluated macroscopically by measuring thelongitudinal length of the colon from the ileocecal junction to the analverge.

Histological Scoring

The entire colon was removed from the cecum to the anus, and the colonlength was measured as marker of inflammation. For histologicalexamination a 2-cm segment of the distal colon was fixed in 10% formalinovernight then in 70% ethanol for paraffin embedding and sectioning.Slides were stained with Harris hematoxylin-eosin (H&E). Histologicalscoring was performed in a blinded fashion by a pathologist, with acombined score for inflammatory cell infiltration (score, 0-3) andtissue damage (score, 0-3). The presence of occasional inflammatorycells in the lamina propria was assigned a value of 0; increased numbersof inflammatory cells in the lamina propria as 1; confluence ofinflammatory cells, extending into the submucosa, as 2; and transmuralextension of the infiltrate as 3. For tissue damage, no mucosal damagewas scored as 0; discrete lymphoepithelial lesions were scored as 1;surface mucosal erosion or focal ulceration was scored as 2; andextensive mucosal damage and extension into deeper structures of thebowel wall were scored as 3. The combined histological score ranged from0 (no changes) to 6 (extensive cell infiltration and tissue damage).

The histology damage score was calculated on a 12-point scale: loss ofarchitecture, 0-3; inflammatory infiltrate, 0-3; goblet cell depletion,0 or 1; ulceration, 0 or 1; edema, 0 or 1; muscle thickening, 0-2; andpresence of crypt abscesses, 0 or 1.

Myeloperoxidase Activity (MPO)

Myeloperoxidase activity in colon tissues was determined as described.Briefly, the colon tissue was rinsed and homogenized in 50 mMpotassium-phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich, St. Louis, Mo.). The homogenatecentrifuged at 14000×g for 15 min and supernatant was used formeasurement of MPO activity normalized on protein concentration.Supernatant was added to 1 mg/ml o-dianisidine hydrochloride(Sigma-Aldrich, ST. Louis, Mo.) and 0.0005% hydrogen peroxide, and thechange in absorbance at 460 nm was measured. One unit of MPO activitywas defined as the amount that degraded 1 mmol peroxide at 37° C. perminute and expressed in units per milligram protein.

Cytokine ELISA

Colonic tissues (50-100 mg) were homogenized in 1 ml of RIPA buffercontaining inhibitors of proteases (Roche), samples were centrifuged at14000×g for 15 min and supernatant were used for determination ofprotein concentration by using Bio-Rad DC Protein Assay kit. Theconcentrations of IL-6, TNF-α, IFN-γ, and IL-1 beta in colon tissue weremeasured by ELISA using commercial BD OptEIA Set kits (BD Biosciences,Sydney, Australia) and normalized to the concentration of total sampleprotein. TFF2 expression by T-cells.

Splenocytes from wild type and transgenic mice were depleted fromerythrocytes and stimulated with PMA (50 ng/ml) and ionomycin (0.5 μM)for 4 h. Brefeldin (1 μl/ml) was added to prevent TFF2 secretion. Cellswere stained for CD3, CD4 and CD8 antigens 30 min on ice, washed twicewith cold PBS buffer, fixed and permeabilized with Cytofix/Cytospermsolution (20 min on ice). Then cells were washed twice in Perm/Washsolution and stained (30 min 4° C.) with raised to C-end of TFF2affinity-purified antibody labeled with Alexa Fluor 488. Rabbitantibodies isolated from preimmune serum and labeled with Alexa Flour488 were used in parallel as isotypic control. Cells were washed twicein PBS with 2% FBS and analyzed for TFF2 expression in CD4+ and CD8+cells gated on CD3+ population.

Flow Cytometry Acquisition and Analysis.

Samples were analyzed on LSRII flow cytometer by using software.Typically list mode for 20,000 events for Gr1+CD11b+ cells in alive-gated mode were acquired. Statistical analysis was done by usingisotype matching controls as a reference. Less than 1% positive cellswere allowed beyond statistical marker in the appropriate control. Thedata files were analyzed using FloJo 5.5.5 software.

Cell Sorting.

Gr1+CD11b+ cells were labeled with PerCP5.5 conjugated Gr1 and APSconjugated CD11b+ antibodies and sorted using sorter FASCAria. Thepurity of sorted cells were more than 95% were used for experiments.

In Vivo Bromodeoxyuridine Labeling

Mice were given 3% DSS water or tap water (control group) for 5 days,and then mice were switched on tap water during next 2 weeks. Mice wereinjected with BrdU intraperitonally (1 mg per 20 g/body weight) 24, 4896 h prior to sacrifice them. Splenic cells were stained for CD11b+,Gr1+ and intracellular BrdU according to manufacturer's instructions.Gr1+CD11b+ cells proliferation in vitro assay. Gr1+CD11b+ cellsproliferation was determined by using BrdU Cell Proliferation Assay Kit(Calbiochem). Splenocytes were obtained from spleen TFF2−/− mice treatedwith DSS on day 19 and Gr1+CD11b+ cells were allowed to bind withantibodies for Gr1 antigen and CD11b marker labeled with PerCP Cy andAPC accordingly and sorted by using sorter FACSAria. Sorted cells werecultured 7 days in complete RPMI 1640 medium (Invitrogen) supplementedwith 10% FCS (HyClone), 50 μM β-mercaptoethanol, 1 mMpenicillin-streptomycin, GM-CSF in concentrations 5 and 10 ng/ml.Recombinant mouse TFF2 was added in concentration as indicated. Cellproliferation was determined by addition of BrdU during the final 22 hof culture accordingly protocol of manufacture (Calbiochem).

Cell Growth Study.

Sorted Gr1+CD11b+ cells were grown at 37° C. in humidified 95% air and5% CO₂ in RPMI-1640 medium supplemented with 10% heat-inactivated fetalcalf serum (HyClone), 2 mM glutamine, 100 U/ml penicillin, 100 μgstreptomycin and GM-CSF at concentration 5 or 10 ng/ml. Recombinant TFF2was added at concentrations as indicated. Cell number was determined byTrypan blue exclusion.

Assessment of Apoptosis

Apoptotic cells were quantified by using annexin V-PE apoptosisdetection kit (BD Pharmigen) accordingly company's protocol. SortedGr1+CD11b+ cells were grown with 5 ng/ml GM-CSF for 7 days. TFF2 wasadded at concentrations as indicated at the beginning of experiment.

Tumor Models.

In the AOM/DSS murine model tumorigenesis was induced as describedpreviously with minor modifications (42). Mice (TFF2−/−, WT and TG, age7-8 weeks) were intraperitonally injected with AOM (10 mg/kg bodyweight) and maintained on regular water for 7 days. Animals were thengiven 3% DSS water for 7 days. Control groups were injected with AOMfollowed only regular water.

For skin tumor model mice were subjected two-step cancerogenesis byusing 9,10-dimethylbenz(a)antracene/12-O-tetradecanoylphorbol-13-acetate(DMBA/TPA) treatment ( ). Mice 7-9 week were shaved 2 days prior ofinitiation of tumorigenesis by single topical application with DMBA (100μg/200 μl in acetone) to the shaved dorsal skin. One week afterinitiation mice were treated with topical application of TPA (2 μg/200μl in acetone) 2 times a week until termination of experiment. Tumorswere counted and measured with a caliper once a week. The number oftumors per mouse with the diameter more then 1 mm were counted and datawere expressed as tumor burden (the number of tumors per mouse) andtumor incidence (the percentage of mice with tumors).

A, tumor multiplicity (average number of tumors per mouse±SE) and (B)incidence (percentage of mice with tumors) in wild-type (∘) andK5.RasGRP1 transgenic (•) mice treated with TPA following initiationwith DMBA. C, wild-type (Wt) and K5.RasGRP1 transgenic (Tg) mice bearingtumors. Pictures were taken at the end of the protocol. D, tumor size(diameter in millimeters) at 17 and 28 wk after initiation with DMBA inboth wild-type (Wt) and K5.RasGRP1 transgenic (Tg) mice. Valuesrepresent the mean±SE of all the tumors in each group (n). *, P<0.05;***, P<0.0001 (Student's t test).

Statistical Analysis

Standard errors and significance by Student's two-tailed t-test werecalculated by using Microsoft Excel software.

Results

TFF2 Protein is Expressed in the Splenic T Cells and Induced by T CellActivation.

TFF2 mRNA expression in the murine spleen and thymus has previously beendemonstrated (13,34,36). In rat lymphoid organs the peptide TFF2 wasdetected by radio-immunological assay due to its very low level and itis has been shown that TFF2 is up-regulated several fold upon LPStreatment. However, the source of cells secreting TFF2 was notidentified. Consequently, western blot analysis of extracts from spleenof wild type mice was performed, using the previously developed andcharacterized rabbit polyclonal antibody to the C-terminal peptide ofmouse TFF2 (43). Immunoprecipitation of mouse splenic extracts using theaffinity-purified rabbit IgG identified TFF2 protein in the spleen as aband that migrates with the same motility as native gastric mouse TFF2(FIG. 1A). As a negative control, splenic proteins immunoprecipitated bypre-immune rabbit IgG were used. TFF2 peptide was also detected inspleen extracts by western blot using a sensitive methodology(SuperSignal West Femto Trial Kit). TFF2 peptide could be detected inthe spleen but not the thymus (FIG. 1B); gastric tissues from wild typeand TFF2 knock out mouse were used as positive and negative controls,respectively (FIG. 1B).

To discriminate which major splenic immune cell subset expressed TFF2,resting splenic cells were fractionated into isolated B and T-cellsubsets, and these subsets were analyzed for TFF2 mRNA bysemi-quantitative RT-PCR analysis. A robust band of amplified TFF2 mRNAwas detected in resting splenic T cells (FIG. 1C, lane T), while aminimal signal at best could be detected in the B cell subset (FIG. 1C,lane B). An increase in circulating trefoil proteins was observed inearlier studies in rats challenged with lipopolysaccharides (LPS) (34).To evaluate the possibility of TFF2 regulation in splenic cells byimmune activation, murine splenocytes were stimulated with T- and B-cellspecific mitogens, concavalin A (Con A) and LPS, respectively. TotalmRNA was isolated from stimulated splenocytes, and the level of TFF2mRNA was analyzed by quantitative real-time PCR. A significant increasein TFF2 mRNA abundance was observed with both treatments (FIG. 1D), witha 2.5-fold increase with LPS and a 40-fold increase with Con Astimulation. Since the increase in TFF2 mRNA expression appeared to bemuch greater after treatment with the T-cell mitogen, the effect ofspecific activation of T cells was examined using anti-CD3 antibodies.Treatment with the anti-CD3ε antibody resulted in a 2.6-2.8-foldincrease in TFF2 mRNA. Thus, the upregulation of TFF2 expression withthe setting of specific T cell stimulation suggests a possible role forTFF2 in T cell function or homeostasis in mice.

It has been long known that trefoil peptides can be overexpressed in thesite of ulceration or mucosal damage (44-47). TFF2 mRNA/proteinproduction can be induced in gastric mucosa as rapidly as 1-4 hfollowing injury (10,48). Therefore TFF2 protein/mRNA expression wasanalyzed in the spleen tissues of wild type and transgenic mice duringadministration of 3% DSS water (FIGS. 1E, 1F, 1G). A noticeable increaseof TFF2 protein was found after 24 h in spleen of both types of mice inresponse of DSS treatment (FIGS. 1E, 1G), as well as an increase themRNA TFF2 level (FIG. 1F). The TFF2 mRNA level was also analyzed insorted T-cells from spleen of mice receiving 3% DSS water.

Transgenic Mice Overexpressing TFF2 in T-Cells Show Attenuated DSSColitis.

To further investigate the role of TFF2 specifically in the immunecompartment in vivo, transgenic mice were created with the enforcedexpression of trefoil peptide in T cells. An expression construct inwhich expression of the murine TFF2 gene (cDNA/ORF) was governed by thehuman CD2 promoter/enhancer (FIG. 2A) was generated. The latter haspreviously been shown to drive a T cell-specific transgene expression inthe thymus and spleen (49,50). The CD2-TFF2 construct was used toproduce 4 transgenic founders lines, identified by PCR-based screen oftail genomic DNA. All four lines showed germline transmission and F1offspring were tested for CD2/TFF2 mRNA expression in the spleen andthymus. Hybrid CD2-TFF2 transcripts (as amplified RT-PCR products of 351bp) were detected in both spleen and thymus of tested pups (FIG. 2B).TFF2 protein overexpression was also detected in the spleen and thymusof transgenic mice (FIG. 2C).

Previously it has been shown that TFF2 knockout mice exhibited anincreased susceptibility to DSS colitis (13). Consequently, thesusceptibility of CD2-TFF transgenic (TG) mice to DSS colitis wasexamined and compared to both WT and TFF2−/− mice. DSS (3%) was givencontinuously in the drinking water and mortality was used as the primaryendpoint. TFF2−/− mice as expected showed a higher mortality ratecompared to WT and TG mice; however, the CD2-TFF transgenic (TG) mice,overexpressing TFF2 only in their thymus and spleen, were more resistantthan wild type mice to DSS treatment (FIG. 3A). In order to examineinflammatory immune response at earlier time points, the same 3 groupsof mice (WT, TFF2−/−, TG) mice were given 2.5% DSS for 5 days andobserved up to day 19. Again, TFF2−/− mice showed a more severeresponse, with a marked reduction in colonic length and enlarged spleenmass by day 19 compared to the two other groups (FIG. 3 B, C, D, E).There were no statistically significant differences between the TG andWT mice; however, the DSS-treated TG spleen remain equivalent to that inuntreated mice, while the spleen of DSS-treated wild type mice wasstatistically larger than that of untreated wild type mice (FIG. 3E).Histological examination of the colon at day 19 revealed inflammation,crypt atrophy, and erosions in all groups of mice, with a trend towardless inflammation in the TG mice compared to TFF2−/− but the differenceswere not statistically significant in this acute colitis model (FIG. 3F,G).

Changes in cytokine expression in the three groups of mice were analyzedafter a single cycle of DSS for 5 days. In WT C57BL/6 mice, this resultsin a progressive chronic colitis characterized by increased levels ofinflammatory cytokines IL-1β, IL-6, INF-γ and TNF-α in colonic tissuesover the time (51-53). In accordance with published data, significantupregulation of mRNA for IL-1β, IL-6, TNF-α and INF-γ in were detectedin WT and TFF2−/− mice compared with untreated wild type mice (FIGS.13A, B, C, D). In contrast, in CD2-TFF2 (TG) mice a statistically lowerlevel of mRNA transcripts for IL-1β, IL-6, TNF-α and INF-γ, and a lowerIL-1β protein level, were observed compared with both wild type and KOmice. TFF2−/− mice also had a statistically significant increase in MPOactivity compare with WT and TG mice (FIG. 4E). Interestingly, TFF2−/−mice with the highest IL-1β mRNA level had the highest MPO activity,while mice with the lowest MPO activity also showed the lowest IL-1βmRNA level. Since IL-1β expression and MPO activity are typicallyassociated with myeloid cells, it is possible that the absence of TFF2in TFF2-deficient mice leads to enhanced mobilization and recruitmentinnate immune myeloid cells.

The IL-2 receptor alpha chain CD25 is a marker of activated of CD4+T-cells (54,55), and previous studies have suggested that expression ofthe IL-2R alpha chain on CD4 T-cells in both the colon and draininglymphatic nodes from the intestine is associated with increased diseaseactivity in experimental model of colitis (56-58). The proportion ofCD4+CD25+ T-cells in TG vs. WT mice after DSS treatment was evaluated;while there was no difference in splenic CD4+CD25+ cells between TG andWT mice at selected time points, the proportion of activated CD4+CD25+cells in the colon and lymphatic nodes was lower in TG mice compared toWT mice at 14 day after DSS treatment (FIG. 14A). Transgenic mice alsohad a lower proportion of colonic activated CD4+CD25+ T-cells at 19 dayafter DSS treatment. Since CD4+CD25+ T-cells expressing Foxp3 areconsidered as a major population of T regulatory cells that suppressinnate and adoptive immune response upon inflammatory change,CD4+CD25+Foxp3 cells were measured in lymphatic nodes in mice WT and TGat 19 day of DSS water treatment. However, the number of CD4+CD25+Foxp3cells was decreased by this time point in both groups of mice,suggesting that the anti-inflammatory effect of TFF2 is likely unrelatedto direct modulation of TReg numbers (FIG. 14B).

Chimaeras Mouse Studies Confirm a Role for Hematopoietic-Derived TFF2 inthe Modulation of Acute Inflammatory Response.

Since TFF2 is expressed in the gastric epithelial cell compartment aswell as the immune compartment, bone marrow transplantation experimentswere carried out to assess further the importance of hematopoieticcell-derived TFF2. wild type mice were lethally irradiated, andtransplanted with bone marrow from TFF2−/−, WT and TG mice. Colitis wasinduced using 5% DSS in the drinking water for 5 days followed byregular water for 19 days. At 6 days, mice transplanted with TFF2−/−bone marrow mice had worse (gross) rectal bleeding, while micetransplanted with bone marrow from TG mice showed a smaller spleen sizeand greater body weight (FIG. 5A, C). By day 19, mice receiving bonemarrow from TFF2−/− mice retained a statistically bigger spleen (FIG.5C) and shorter colon (FIG. 5B) compared with the other two groups. Inaddition, mice that received TFF2−/− bone marrow showed a higher proteinlevel of IL-1 beta relative to the other two groups (FIG. 5E). Incontrast, mice that received bone marrow from transgenic mice displayedan amelioration in their colitis, including a greater body weight,normal spleen mass and colon length (FIG. 5A, B, C, D). On histologicalexamination, mice receiving TFF2−/− bone marrow had a greater degree ofmucosal injury with gross infiltration of immune cells in submucosa(FIG. 5G), however these differences did not reached statisticalsignificance (FIG. 5H). The fact that wild-type mice transplanted withbone-marrow from TFF2-deficient mice had an overall stronger colitisphenotype (such higher body loss, gross bleeding, shorter colon, largerspleen, higher level of IL-1β) as compared to those transplanted withbone marrow from wild-type or transgenic mice revealed protective effectof bone marrow derived TFF2 in colonic homeostasis.

In order to determine whether TFF2-expressing bone marrow could rescuethe TFF2-deficient phenotype lethally-irradiated TFF2-deficient micewere transplanted with bone marrow derived from WT, TG or TFF2−/− mice.The transplanted mice were studied after exposure to DSS at a slightlylower concentration (2.5%). Mice receiving bone marrow fromTFF2-deficient mice showed higher body weight loss at day 4 and 5 thenmice received bone marrow from transgenic mice, however, there was not adifference compared to mice with bone marrow transplanted from wild-typemice (FIG. 6A). In addition, mice receiving bone-marrow from WT or TGmice showed less severe diarrhea, lower colonic MPO activity and alonger duration of rectal bleeding than mice receiving TFF2−/− bonemarrow (FIGS. 6B, C, G). On day 19, the chimaeras with bone marrow fromTFF2−/− mice still showed the traces of blood in feces, displayedshorter colon and larger spleens compared with the other two groups(FIGS. 6C, D, E). Thus, transplantation of bone marrow from wild-type orCD2-TFF2 transgenic mice into TFF2-deficient mice can partially rescuethe inflammatory phenotype.

Finally, the relative contribution of epithelial-derived TFF2 versusbone marrow-derived TFF2 on survival rate when DSS (5%) was continuouslyadministered in the drinking water were compared. ChimaerasTFF2-deficient mice received bone marrow from knockout or wild type ortransgenic mice died faster then chimaeras wild-type transplanted withbone marrow from wild type or transgenic or knockout mice uponcontinuous treatment with 5% DSS (FIG. 6H, upper and lower panel).Without being bound by theory, these experiments showed that TFF2expressed in epithelial cells of the GI tract likely has a greaterprotective effect compared with TFF2 from non-epithelial cells.

DSS Treatment Results in Greater Accumulation of Myeloid Cells in Spleenin TFF2-Deficient and Wild Type but not in Transgenic Mice.

While the data above indicated some degree of protective effect fromhematopoietic-derived TFF2 on colonic inflammation, it was also noted inthe chimeric studies that TFF2-deficiency was associated with a largerspleen size, while CD2-TFF2 transgenic mice exhibited a lower spleensize after DSS colitis. Under normal conditions, less than 4% of cellsin the spleen are myeloid cells expressing CD11b+Ly6C+(59), but duringthe chronic phase following DSS treatment, the WT spleen enlarges with asignificant increase in the total number and proportion of myeloid cells(51,60). Consequently, the possibility that the increase in spleenweight in the setting of TFF2 deficiency might result from the expansionof Gr1 CD11b+ cell population was investigated. Histological examinationof heamotoxylin/eosin stained spleens on day 19 after DSS treatmentrevealed that cells with the typical ovoid or circular nucleus,characteristic of immature myeloid cells (IMC), were much more abundantin TFF2−/− and WT mice than in CD2-TFF2 transgenic mice (FIG. 15). Inorder to determine the cause for this expansion of IMCs, the spleensfrom TFF2−/−, WT and TG mice of SPKO mice were stained intensively forproliferative marker Ki67. Increased proliferation was observed inTFF2-deficient mice, while decreased proliferation was seen in TG mice(FIGS. 7A, B). Staining of serial sections for Ki67 and Gr1 antigensrevealed that TFF2−/− mice showed significantly more activelyproliferating Gr1+ cells than WT mice, while TG mice showed fewerproliferating Gr1+ cells than WT mice. Importantly, staining for Ki67and Gr1 antigens was found in the red pulp zone where myeloid cellstypically reside (FIGS. 7A, B). These observations suggested thatsplenomegaly in DSS-treated TFF2−/− mice indeed might associate withexpansion of immature myeloid cells likely due to intensiveextramedullary hematopoiesis.

To further explore the role of TFF2 in the modulation of splenocytes,changes in myeloid cells were examined by FACS analysis over the courseof DSS-treatment using antibodies to CD11b and Gr-1. TFF2-deficient miceaccumulated the highest proportion of CD11b+Gr1+ cells, while CD2-TFF2transgenic mice showed the lowest percentage (FIG. 7D, FIG. 16A, B). Theproportion of CD11b+Gr1+ cells increased over time in TFF2−/− and wildtype mice with a peak on day 19, in contrast to the CD2-TFF2 transgenicmice which showed an insignificant increase in proportion of these cellsat all time points (FIG. 7D, left). The absolute number of doublepositive CD11bGr1 cells significantly increased in TFF2−/− and to alesser extent in wild type mice, while in transgenic mice there was muchless accumulation of IMCs (FIG. 7C-E).

Changes in the numbers of Gr1+CD11b+ cells in the bone marrow over thecourse of DSS-induced colitis were investigated. An increase inGr1+CD11b+ cells in bone marrow of all groups of mice was found; howeverTFF2−/− mice showed the highest percentage of IMSc while the TG miceexhibited the statistically lowest percentage of Gr1+CD11b+ cells (FIG.7D, right).

Recently it has been proven that myelopoisis in spleen significantlycontributes in accumulation of myeloid cells under various conditionsincluding injury and cancer. Gr1+CD11b+ cells comprise heterogeneouspopulation including immature macrophages, dendritic and myeloid cellsat various stages of differentiation as well their precursors (61)Almand, 2001). Expansion of myeloid cells in spleen was likely as aresult of both recruitment from bone marrow and local proliferationtheir precursors in spleen of tumor-bearing mice ((62) Cortez-Retamozoet al., 2012) and in case of myocardial infracture.

To determine whether the accumulation of splenic Gr1+CD11b+ cellsoccurred due to active proliferation in periphery BrdU was injected inmice to label in vivo dividing cells. After 96 h splenic cells wereisolated and stained for CD11b+, Gr1+ and intracellular BrdUincorporation was detected according to manufacturer's instructions. Itwas observed that splenic cells stained for Gr1 and CD1b markersactively proliferate during recovery phase of DSS-induced colitis,however cells from TFF2-deficient mice display higher BrdU uptake thenother two groups (FIG. 16A, B). Approximately 66% Gr1+CD11b+ cellsincorporated BrdU in TFF2−/− mice, in WT mice around 38% of Gr1+CD11b+cells were positive for BrdU uptake, while less then 8% of Gr1+CD11b+cells were positively stained for BrdU (FIG. 7E).

Since granulocyte/macrophage precursors may give rise to myeloid cellpopulation and contribute to their expansion in spleen (63) Leuschner Fet al., 2012) colony-forming capacity of splenocytes obtained from allgroups of mice on day 19 after DSS treatment was evaluated by usingmedium MethoCult M3434. This medium support growth of erythroid,granulocyte-macrophage and multi-potential granulocyte, erythroid,macrophage, megakaryocyte progenitors (StemCell Technology). Splenocytesfrom TG mice showed the lowest capacity to form colonies, whilesplenocytes from TFF2−/− deficient mice formed significantly morecolonies compared with TG and WT mice accordingly (FIG. 7F). Takentogether these data suggest that TFF2 suppresses the expansion ofGr1+CD11b+ cells and their precursors in vivo.

TFF2 Suppresses the Proliferation and Growth of Gr1+CD11b+ Cells inEx-Vivo Culture.

To further explore the effect of TFF2 on Gr1+CD11b+ cells these cellswere characterized morphologically and phenotypically. BecauseGr1+CD11b+ cells comprise heterogeneous population including immaturemacrophages, dendritic and myeloid cells at various stages ofdifferentiation (Almand, 2001) splenocytes were labeled with antibodiesagainst antigens Gr1 (which recognizes both monocytic and granulocyticmarkers) and CD11b then sorted by using sorter FACSAria. Sorted doubleGr1+CD11b+ cells with purity around 95% (FIG. 9C) representheterogeneous population of cells with ring shaped and segmented nuclei(FIG. 9A) and thus show morphological characteristic consistent withphenotype of immature myeloid cells. These cells displayed antigenspreviously reported being expressed on immature myeloid cells suchearlier marker of myeloid progenitors CD31, CD115, macrophage antigenF4/80 and dendritic cell marker CD11c, (FIG. 9B) (64-66; Venkatesh L etal., 2010). They also were positive for monocytic marker Ly6C (FIG.59C-D, FIG. 61B). These cells are premature as antigen-presenting cellsbecause they express low levels of MHC class II, co-stimulatorymolecules CD86, CD80 and CD40. Sorted Gr1+CD11b+ cells grow, loosemarker Gr1, decrease expression of macrophage marker F4/80 but increaseexpression of dendritic cell marker CD11c over 7 days in presence ofGM-CSF (10 ng/ml). Next it was investigated whether TFF2 directly affectthe growth of sorted Gr1+CD11b+ cells in vitro culture. BecauseGr1+CD11b+ cells died in absence of growth GM-CSF, the effect of TFF2 inpresence of low concentration of GM-CSF in medium after 7 days ofculture was analyzed. GM-CSF is well-known factor that support viabilityand differentiation of Gr1+CD11b+ cells (Morales J K & Kmieciak M,Breast Cancer Res Treat 2010). In the initial series of experimentsGr1+CD11b+ cells were cultured with 10 ng/ml GM-CSF only and withaddition of TFF2 in parallel setting. Since there was no difference inthe number of viable cells and BrdU uptake by Gr1+CD11b+ cells under theabove conditions the concentration of GM-CSF was lowered to 5 ng/ml. Thenumber of viable cells decreased in dose-dependent manner upon TFF2supplementation in the range 0.2 μM-4 μM (FIG. 10A). Addition of TFF2also resulted to a decreased BrdU uptake by Gr1+CD11b+ cells indose-concentration manner (FIG. 10B). However, recombinant TFF2 does notseem to induce apoptosis of Gr1+CD11b+ cells even in higherconcentrations.

Expression of TFF2 by Splenic T Cells Suppresses the Development ofColon and Rectal Tumors Following AOM/DSS Treatment

Gr1+CD11b+ cells comprise several populations including cells withimmunosuppressive function (MDSC). These cells are induced by variousconditions including inflammation and inhibit tumor immunity in studieson mouse tumor models and cancer patients (1,67,68). Because TFF2−/−mice developed worse colitis with higher accumulation of Gr1+CD11b+cells in bone marrow and spleen compare with wild type and transgeniccounterparts it is possible TFF2−/− mice were more likely to developtumors in cancer models associated with inflammation while transgenicmice would be resistant then wild type and TFF−/− deficient mice.Knockout, wild type and transgenic mice were injected with singleintraperitoneal injection of procarcinogen azoxymethane (AOM) following3% DSS regiment during next 7 days. Mice were analyzed for the presenceof colon tumor five months later. Both wild type and TFF2−/− micedeveloped tumors in the third part of distal colon, howeverTFF2-deficient mice clearly showed higher number of small tumors withsize 1-3 and 4 mm (FIG. 11A, B). Transgenic mice showed no tumor at allor only a single tumor, depending on experiment 70-90% of transgenicmice do not develop tumor. Histological examination showed largeradenomas with increased inflammatory cell infiltration in colonictissues of TFF2−/− deficient mice compare with wild type counterparts(FIG. 11C). Higher tumor burden in TFF2−/− mice correlates with anincreased proportion of Gr1+CD11b+ cells in the spleen and bone marrowcompare with wild type and transgenic mice (FIG. 11D). Transgenic micedisplayed the same proportion of Gr1+CD11b+ cells in bone marrow as wildtype, but lower in the spleen and in blood (FIG. 11D). Very recently ithas been shown that tumor induces splenic accumulation of hematopoieticstem cells (HSCs) and lineage progenitors cells such GMP and theirimportant contribution in tumor progression (62,68). In order to testthe presence of these populations in spleen and bone marrowcolony-forming units assay and flow cytometry with markers and gatingstrategy described earlier (62) (FIG. 40) was performed. TFF2-deficientmice showed a greater proportion of GMP and higher number of CFU comparewith WT and TG mice. Thus TFF2 suppresses the expansion ofgranulocyte-macrophage progenitors in vivo and therefore inhibitscancerogenesis. Interestingly, suppressive TFF2 effect on tumor growthin syngenic model, when tumorigenesis has been initiated by subcutaneousinjection of EL-4 cells without application additional inflammatorystimuli was not found. It is well known that the AOM/DSS modelassociates with colonic inflammation with increase of IL-b, Il-6, TNFalpha cytokines in colon tissues. Indeed, TFF2−/− deficient mice showedsignificantly higher expression of mRNA for IL-1b and IL-6 in tumortissues compare with tumor WT and TG mice (FIGS. 11F, G). TFF2−/−deficient mice also showed higher fold change of IL-6 mRNA level incolon tissues uninvolved in tumor compare with other two groups, but thesame increase mRNA for IL-1b as in wild type mice. In parallel thelowest increase of mRNA for IL-1b and IL-6 was in colon tissuesuninvolved in tumor in transgenic mice (FIGS. 11H, I). Since IL-1b hasbeen shown to promote expansion of myeloid cells these data areconsistent with higher tumor incidence in TFF2−/− deficient mice.

From the obtained data, and without being bound be theory, TFF2attenuates tumor incidence by limiting the number of Gr1+CD11b+ cells insite of tumor.

Discussion

Protective role of TFF2 on stomach and colon is well documented andattributed to gastric origin, where its abundant expression is observed.Earlier studies showed that gastric TFF2 increased the viscosity ofmucus which covers and protected cell epithelium and also promotedregenerative process by stimulating migration epithelial cell to placesof injury (9,10,69-71).

However, trefoil peptides were detected in immune organs and functionalrelevance from immune compartment is still not fully understood. Severalstudies suggest that TFF2 is a negative regulator ofgastrointestinal/systemic inflammation and immune cytokine response(13,36). Indeed, recent study showed that TFF2 promotes type 2 immunityby releasing IL-33 from epithelial, macrophages and dendritic cellsthrough CXCR4 receptor (39). This process is beneficial in parasiticinfection but pathogenic in context of asthma. In the example presentedherein, splenic T-cells were identified as a source of immune cellsexpressing TFF2 under normal physiological conditions in spleen. Forfurther analysis mice overexpressing TFF2 under human CD2 promoterspecifically in T-cells were created and their inflammatory response wasexamined in a model of oral DSS administration which results in directcolonic injury to intestinal epithelial cells and inflammation due toincreasing proinflammatory stimuli (72).

In accordance with earlier published data, results presented herein maynot be explained by known barrier and reparative mechanism carried outby gastric TFF2. First, chimaeras WT with TFF2-deficiency in immunecompartment showed higher susceptibility to DSS-treatment despite thepresence of TFF2 in gastric epithelial cells. Second, chimeras TFF2-nullmice with expression of TFF2 only in immune compartment showed lessinflammation compared with TFF2-null mice. Thus TFF2 expressed byT-cells exhibits some dampening effect on colonic and systemicinflammation. Remarkably, protective effect was observed despite thefact that quantity of TFF2 in immune compartment is significantly lowerthan secreted by gastric epithelial cells or recombinant TFF2 used fortreatment in earlier reports.

Experiments in the in vivo model indicate that TFF2 besides knownbarrier and reparative function in gastrointestinal tract also isinvolved in anti-inflammatory mechanism provided by T-cells expressingTFF2. Exacerbated immune response in TFF2−/− mice was observed inaccordance with previous data and lower systemic inflammatory immuneresponse in transgenic mice compare with wild type mice in DSS model. Atransient significant increase in the number of Gr1+CD11b+ cells wasfound in TFF2−/− with less expansion in wild type mice, while onlymoderate accumulation of these cells was observed in transgenic mice. Ithas been widely accepted that chronic intestinal inflammation isgenerally associated with expansion of colitogenic T-cells. Howeversignificant increase of Gr1+CD11b+ cells also has been reported underexperimental conditions of chronic gut inflammation induced with DSS(51,73) or T-cell adoptive transfer model of chronic colitis (Haile L Aet al., Gastroenterology, 2008; Ostanin D et al., I Immunol., 2012). Inthese mouse models chronic colitis was associated with of accumulationof immature myeloid Gr1+CD11b+ cells similar to those described intumor-bearing mice. While it is long established that IMC fromtumor-bearing mice exhibit suppressive function (MDSC) and contribute incancer progression, the role of IMC cells in pathogenesis ofinflammatory bowel disease is still not clear. Some reports suggest thatneutrophils suppress inflammation (Kuhl A A et al, Gastroenterology,2007; Haile L A et al., Gastroenterology, 2008; Nemoto Y et al,Inflammation Bowel Dis. 2008; Zhang R et al., Inflamm Allergy DragTargets 2011) but other studies do not support this conclusion (Natsui Met al., J. Gastroent. Hepatol., 1997; Qualls J E et all. J Leuco. Biol2006; Ostanin D et al, J Immunol, 2012). Latest report suggests thatGr1+ neutrophils isolated from colitic mice induce proliferation of CD4+T cells and enhance the production of proinflammatory cytokines byactivated CD4+ T cells perpetuating gut inflammation (Ostanin et al., JImmunol, 2012).

Importantly, expansion of IMC in the experiments accompanies with anincrease of Ki67 marker proliferation in spleen and in vivo BrdU uptakeby splenic Gr1+CD11b+ cells suggesting on much higher extramedullarcells proliferation in TFF2-deficient mice compare with other twogroups. Splenocytes from TFF2−/− mice treated with DSS form morecolonies on medium supporting granulocyte, macrophage, megacaryocyte anderythroid precursors then splenocytes from wild type and transgenicmice. In addition recombinant TFF2 directly suppresses Gr1+CD11b+ cellsproliferation in vitro culture, supporting in vivo data.

Expansion of myeloid cells in spleen due to increase in their turnoverhas been noticed earlier under other pathological conditions suchthermal injury (Noel J et al., 2005). The idea that spleen may be as asource of immature myeloid cells due to active extramedullarhematopoiesis came from observation on massive accumulation of thesecells in spleen of tumor-bearing mice (Johnson J R et al., 1985, Int. JCell Cloning; Serafini P. et al., 2004, Cancer Immunol Immunoter; du'PreS A et Hunter K W Jr, Exp Mol Path., 2007). An expanded red pulp withmegacaryoblasts and metamyeloblasts and reduction of white pulp area inspleen of tumor-bearing mice suggest on extramedullar hematopoiesis(du'Pre S A et Hunter K W, 2007). So called leukemoid reactionscharacterized by splenomegaly due to massive granulocytic infiltrateshave been also reported in human cancers and are associated with a poorprognosis (Sato K et al., J. Urol., 1994; Kasuga I et al., 2001; Nimieriet al., 2003, Annal Hematol; Schniewind B et al., 2005, Cancer Biol.Ther.) However, studies on syngenic tumor model suggest that expansionof IMC in spleen during cancer progression occurs as a result ofproliferation and differentiation of these cells in bone marrow andsubsequent migration from bone marrow to the blood but not due toproliferation in spleen (Ueha S et al., 2011). Indeed, by using aparabiosis system and in vivo BrdU incorporation these authors showedthat immature myeloid cells have proliferated primarily in the bonemarrow and not in peripheral tissues (Sawanobori Y et al., Blood, 2005).From earlier experiments it has been suggested that myeloid cellsundergo extramedullar proliferation in response to soluble tumor-derivedfactors (Young M R, Young M E, Cancer Res., 1988; Kusmartsev S A, Li Y,J. Immunol., 2000). Recently it has been shown that spleen becomereservoir of monocytes/macrophages that mobilize and migrate to inflamedtissue in response to myocardial infraction-induced heart injury andparticipate in wound healing (Swirski et al., 2009; Leuschner F et al.,2012). Later it has been shown that spleen also contributes inflammatorymonocytes to atheroma in atherosclerosis (Robbins C S et al.,Circulation, 2012) and monocytes/granulocytes in tumor sites duringcancer progression (Cortez-Retamozo V et al., 2012).

Recent studies on RAG−/− mouse model clearly showed that chronic colitisis accompanied by the massive infiltration of myeloid cells in laminapropria and also associated with dramatic myelopoiesis with around 10folds more myeloid cells (primary neutrophils) than T-cells in spleen(Ostanin D et al., J Immunol., 2012). Moreover, these infiltratedimmature myeloid cells acquire the phenotype and function of APC withinthe inflamed bowel and contribute to disease progression. Therefore itis possible that some conditions such injury, inflammation and tumorgrowth stimulate extramedullar hematopoiesis in spleen that become anadditional source of IMC which may contribute to outcome of disease.Although it has not been found directly how spleen-derived IMCcontribute in cancer progression based on presented data it seems TFF2to be the factor that suppresses proliferation of Gr1+CD11b+ cells inspleen and this accounts on less tumor incidence in cancer modelsassociated with inflammation.

Anti-tumor activity of TFF2 was evaluated in two types of cancer modelsassociated with inflammation: in colon cancer model initiated by AOMfollowing DSS treatment and in skin cancer model initiated by DMBAfollowing TPA treatment. In the first model it was found that TFF2−/−deficient mice are more susceptible while TG mice are more resistant totumorigenesis. what is consistent with their higher susceptibility toinflammation and higher proportion of inflammatory Gr1+CD11b+ cellsobserved for these mice upon DSS treatment.

Extensive studies on mechanisms by which MDSCs exert theirimmunosuppressive function have been done as well factors that promotetheir expansion have been revealed. Attention should be dedicated topoint which factors restrict expansion of MDSC.

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Example 6 Splenic T-Cell Derived TFF2 Inhibits InflammatoryCarcinogenesis Through Suppression of Immature CD11b+Gr1+ Myeloid Cells

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous populationof CD11b+Gr1+ cells that expand during cancer and can contribute toneoplastic progression. Trefoil factor 2 (TFF2), a small proteaseresistant protein expressed by the gastric epithelial cells and splenicT cells, can function in part as an anti-inflammatory peptide. Here, itis shown that in response to carcinogenic stimuli, TFF2 is upregulatedin memory T cells in the spleen through a vagal neural circuit andfunctions to suppress proliferation of myeloid progenitor cells.Knockout of the TFF2 gene leads to an expansion of MDSC, and markedacceleration of tumor growth in response to AOM/DSS treatment. Moreover,overexpression of TFF2 in splenic T cells completely suppressed bothMDSC expansion and colon cancer induction. Suppression of CD11b+Gr1+cellular expansion by TFF2 correlated with an increase in CD8+ T cellsin response to AOM/DSS colon carcinogenesis. In vitro studies showedthat the effect of TFF2 involved a direct suppression of proliferationby granulocyte-macrophage progenitors (GMP) and CD11b+Gr1+ immaturemyeloid cells (IMC). Bone marrow transplant studies confirmed the roleof hematopoietic TFF2 expression in inhibition of the cancer phenotype.Taken together, these studies validate the role for CD11b+Gr1+ cells inearly cancer progression, and point to a possible therapeutic role forTFF2 in suppression of MDSCs and cancer.

Introduction

Tumor growth and progression can be accompanied by expansion ofmyeloid-derived suppressive cells (MDSCs), immune cells characterized inmice by the co-expression of surface markers Gr1 and CD11b (A1). MDSCsare a heterogeneous population of immature myeloid cells that accumulatein the bone marrow, spleen and peripheral blood of tumor-bearing mice,and can be elevated up to ten-fold in the blood of patients with diversetypes of cancer. The accumulation and activation of MDSCs can occur inresponse to factors secrete by tumors, such as VEGF, GM-CSF, IL-1β, IL-6and PGE2 (A2-A5, A6, A7, A8, A9, A10), which can also be increased inthe setting of chronic inflammation. These carcinogenic and/orinflammatory factors can result in the expansion of MDSC throughstimulation of myelopoiesis and inhibition of myeloid celldifferentiation.

Expansion of CD11b+Gr1+ myeloid cells can also occur following trauma,infection and acute inflammation, but in cancer these cells persist andcan acquire the profound ability to suppress T cell activation throughmultiple mechanisms (A1). Thus, the sustained expansion of MDSCs withimmunosuppressive ability that can be seen in cancer is typically absentin acute inflammation. Resolution of inflammatory responses can bemediated by endogenous anti-inflammatory factors secreted by host immunecells in response to inflammatory signals (A2-A4). One anti-inflammatorypathway, termed the inflammatory reflex involves a neural reflex,whereby stimulation of the vagus nerve can activateacetycholine-synthesizing memory (CD4+ CD44^(hi)) CD69L^(lo)) T cells,which can inhibit cytokine release and attenuate inflammation-mediatedinjury (A5). These observations suggest the possibility that failure ofthese reflex anti-inflammatory mechanisms, which can normally limit theexpansion of myeloid cells, can contribute to nonresolving inflammationand cancer.

The central nervous system can regulate the innate immune responses viathe vagus nerve, a mechanism termed the cholinergic anti-inflammatorypathway. Vagus nerve stimulation can inhibit proinflammatory cytokineproduction by signaling through the alpha7 nicotinic acetylcholinereceptor subunit expressed on macrophages, lymphocytes, neurons andother cells. The mechanism is called the inflammatory reflex.Administration of nicotine, an alpha7 agonist that mimics vagus nervestimulation, can increase proinflammatory cytokine production andlethality from promicrobial sepsis in splenectomized mice, indicatingthat the spleen can be a major contributor to the anti-inflammatoryeffect via the cholinergic pathway.

Trefoil factor 2 (TFF2) is a secreted peptide that can function as ananti-inflammatory peptide. TFF2 is a member of the trefoil factor family(TFF), which in mammals includes three secreted proteins (TFF1, TFF2,and TFF3), each of which possesses a highly conserved triple loopstructure (the trefoil domain) and are expressed in the gastrointestinaltract. TFF2, similar to other trefoil proteins, can play a role inmucosal repair and the maintenance of mucosal integrity throughinteractions with epithelial cells (A6-A8). However, TFF2 can alsofunction as an anti-inflammatory peptide. TFF2-deficient mice atbaseline show a minimal phenotype, but in response to DSS showed delayedhealing and recovery (A14, A15). Studies have suggested that TFF2 caninfluence leukocyte migration, recruitment or responses (A10, A13,A16-A19). It has been shown that splenic T cells from TFF2-deficientmice are hyper-responsive to IL-1β stimulation, and that TFF2 canmodulate signaling through CXCR4 and induce the release of IL-33 fromlung epithelial, dendritic cells and macrophages, thus promoting a Th2type immune response (A15, A20-22). The recognition that TFF2 can play abroader role in immune responses beyond the gastrointestinal tract wassupported by the observation that TFF2 mRNA expression can be detectedat low levels in rodents primary and secondary lymphatic organs (thymusand spleen), where expression was increased upon LPS treatment (A15,16).

In addition, several studies have shown that TFF2 can be downregulatedin cancer and may function as a tumor suppressor gene. Loss of TFF2 hasbeen observed during the progression of human intestinal-type gastriccancer (A23, A24), and it has been shown that TFF2 expression isdownregulated due to aberrant promoter methylation (A25, A26) in thesetting of H. pylori infection. Moreover, TFF2-deficient mice progressmore quickly to dysplasia in inflammatory models of gastriccarcinogenesis (A26, A27).

In this example, it is shown that TFF2 is expressed in memory T cells,regulated by a vagal nerve circuit, and can function to suppressmyelopoiesis in the spleen in response to inflammatory stimuli.Importantly, it is shown that TFF2 can function to suppress thedevelopment of myeloid-derived suppressor cells (MDSCs) in response tocarcinogens, and that overexpression of TFF2 can markedly suppressgastrointestinal tumorigenesis.

Results

TFF2 can be Expressed in the Splenic CD4+ Memory T Cells and Regulatedby the Vagus Nerve

While previous studies revealed that TFF2 mRNA can be expressed in therodent in spleen and regulated by inflammatory signals (A15, A16, A18),the precise cellular origin of TFF2 was not identified. Antibodies tothe TFF2 C-terminus (A15) identified TFF2 protein in whole spleen as aband at the position similar to those detected in stomach of wild typemice by western blot (FIGS. 1A-B). Fractionation of resting spleniccells revealed higher TFF2 mRNA in T cells compared to B cells (FIG.1C). Consistent with this finding, stimulation of murine splenocytesshowed a 40-fold increase in TFF2 mRNA abundance with a T-cell specificmitogen (concavalin A) compared with a 2.5 fold increase with a B-cellspecific mitogen (LPS) (FIG. 1D).

Given previous studies suggesting a protective role for trefoil peptidesin rodent colitis models, and the upregulation of TFF2 in the gut inresponse to injury (A28-A31) (A7, A32, A33), TFF2 protein expression wasanalyzed in the whole spleen of wild type mice during administration of3% DSS water, a marked increase was observed after 24 hours of DSS, withcontinued expression through day 19 (FIGS. 1E, 1G). Similar increaseswere observed in TFF2 mRNA expression in the DSS model.

Stimulation of the vagus nerve can inhibit cytokine release and candownregulate systemic inflammation through interactions with memory CD4+T cells, (A34-A36). TFF2 can also be part of an anti-inflammatorypathway, thus TFF2 expression in the same subset of memory CD4+ T-cellsin spleen was analyzed (FIG. 18). Analysis of flow sorted spleniclymphocytes from DSS-treated mice revealed that memory CD4+CD44^(high)CD62L^(low) T-cells expressed 8-fold higher TFF2 mRNAcompared with unsorted total CD4+ T-cells (FIG. 19). Next, TFF2 mRNAlevels were analyzed in spleens from mice with bilateral truncalvagotomy with pyloroplasty (VTPP) before and after 6% DSS treatment for2 days compared with control mice without vagotomy. While VTPP did notalter TFF2 mRNA levels in spleen in untreated mice (FIG. 21), theincrease in TFF2 mRNA levels following vagotomy was completely abrogatedfollowing vagotomy (FIG. 20). Taken together, these findings suggestthat TFF2 can act as an anti-inflammatory peptide expressed in memory Tcells and regulated by the vagus through the inflammatory reflex.

Transgenic Mice Overexpressing TFF2 in T-Cells Display Attenuated DSSColitis.

Given the upregulation of TFF2 in response to inflammation, and itssuggested role as an anti-inflammatory peptide, the ability of TFF2overexpression in the immune compartment to suppress acute or chronicinflammation was tested. Transgenic mice that overexpress murine TFF2specifically within T cells were generated using a well-established CD2promoter construct (FIG. 2A) (A37, A38). TFF2 mRNA and protein wasdetected in the spleen and thymus of CD2-TFF2 F1 offspring (FIGS.2B-2C). CD2-TFF2 transgenic mice appear phenotypically normal and showedno difference in the proportion of T and B-subsets in spleen and T-cellssubsets in thymus. TFF2-null mice exhibited greater susceptibility tocolitis following exposure DSS (A15), with increased spleen size (FIG.28), a marked reduction in colon length (FIG. 3B), increased MPOactivity (FIG. 4E), and a higher mortality rate (FIGS. 3A, 27) comparedto other groups. However, CD2-TFF2 transgenic mice did not statisticallydiffer from wild type mice, although there was a slight trend towardsincreased survival. There was no difference in body weight change afterone cycle of DSS between all groups. Nevertheless, CD2-TFF2 transgenicmice did show lower levels of IL-1α in their colons compared to WT micein the DSS model, and IL-1β levels were even higher in TFF2−/− mice(FIG. 4A). Histological examination of the colon at day 19 revealedinflammation, crypt atrophy, and erosions in all groups of mice, with atrend toward less inflammation in the CD2-TFF2 transgenic mice (FIG.3G).

Since TFF2 can be expressed in the gastric epithelium as well asT-cells, bone marrow transplantation experiments were performed toassess the importance of hematopoietic derived TFF2. Lethally irradiatedwild type mice were transplanted with bone marrow from TFF2-null,wild-type and CD2-TFF2 mice; colitis was induced using 3% DSS in thedrinking water for 5 days followed by regular water, and animals wereassessed on day 19. Transplantation with CD2-TFF2 bone marrow, comparedto WT bone marrow, resulted in attenuated colitis as revealed by greaterbody weight, normal spleen mass and colonic length; in contrast, micetransplanted with TFF2-null bone marrow, compared to WT bone marrow,showed significantly greater loss of body weight, larger spleens,shorter colons and higher levels of IL-1β (FIGS. 5A-5C, 5E). Takentogether, these results suggest a role for hematopoietic-derived TFF2 inmodulating acute inflammation.

Overexpression of TFF2 by Splenic T Cells can Suppress the Developmentof AOM/DSS-Induced Colon and Rectal Tumors

Previous studies have suggested that TFF2 is a tumor suppressor gene.The influence of overexpression of TFF2 on the development of colon andrectal cancer was tested. TFF2-null, wild type and CD2-TFF2 groups weresubjected to the AOM/DSS regimen, and colonic tumors were quantifiedfive months later. Both wild type and TFF2-null mice developed tumors inthe third part of distal colon, with the TFF2-null mice showing thegreatest tumor load (FIGS. 11A-B). TFF2-null mice showed an averagetumor burden that was 3× the tumor burden in WT mice (P=0.0002). Most ofthe of CD2-TFF2 mice (approximately 80-100%) were tumor free, and noanimal in this group developed more than a one lesion. The average tumorburden in CD2-TFF2 transgenic mice was <1 (p-0.004).

Histological examination of adenomas from TFF2-null mice revealedgreater degrees of dysplasia, and increased inflammatory cellinfiltration in colonic tissues of TFF2-null mice compared with wildtype counterparts (FIG. 11C). The inflammatory cells in the colonictumors were a heterogeneous population of leukocytes, includingCD11b+Gr1+ cells with majority of granulocytic CD11b+Ly6G+Ly6C^(lo)subset (FIGS. 26A-B). The greater tumor burden in the TFF2-null groupcorrelated with an expanded CD11b+Gr1+ cell population in the spleen,bone marrow and blood (FIG. 11D). CD2-TFF2 mice showed the sameproportion of CD11b+Gr1+ cells in the bone marrow as wild type mice, buta significantly lower proportion of these cells in the spleen andperipheral blood. Consistent with the increase in colonic myeloid cells,there was increased IL-0 mRNA levels in colonic tumors from TFF2−/−deficient mice compared to colonic tumors from wild type and CD2-TFF2mice (FIGS. 11F, 58, 59A-B), while IL-β (and IL-6) mRNA levels werelower in tumors from CD2-TFF2 transgenic mice. Accumulation ofCD11b+Gr1+ cells in tumors correlated with highest IL-1β level in tumorsite compare with colonic tissues without visible tumor (FIG. 59C). Ashas been reported earlier splenic CD11b+Gr1+ cells consist of twopopulations expressing Ly6G and Ly6C markers with majority ofgranulocyte antigen Ly6G (FIG. 59D).

TFF2 Inhibits Cancer Through Suppression of MDSCs.

The increase in tumorigenesis seen in the TFF2−/− mice was associatedwith splenomegaly and accumulation of CD11b+Gr1+ myeloid cells, whilethe suppression of tumors in CD2-TFF2 transgenic mice correlated with alack of splenic enlargement. DSS-induced colitis has previously beenassociated with splenic enlargement and a significant increase inCD11b+Gr1+ cells (A39-A41). While under normal physiological conditions,TFF2−/− mice and CD2-TFF2 transgenic mice showed normal spleen size andproportions of CD11b+Gr1+ cells in spleen and bone marrow, in responseto DSS, TFF2-null mice showed significantly more Ki67+ proliferatingGr1+ cells than wild-type mice, while CD2-TFF2 mice showed fewer Ki67+proliferating Gr1+ cells compared to WT mice (FIGS. 7A-B). Importantly,staining for Ki67 was most abundant in the red pulp zone where myeloidcells reside. In response to DSS-treatment, the proportion of CD11b+Gr1+cells in the spleen and bone marrow increased over time in TFF2-null andwild type mice with a peak occurring on day 19, whilst in the CD2-TFF2transgenic mice there was a minimal increase in this myeloid lineage(FIGS. 7C, 60).

BrdU labeling studies of DSS treated mice revealed that CD11+Gr1+myeloid cells in the spleens of WT mice proliferate during the recoveryphase of DSS-induced colitis; however, splenic IMC's from TFF2-deficientmice showed greater BrdU uptake than the other two groups (FIG. 39). Upto 7% of CD11b+Gr1+ cells incorporated BrdU in TFF2-null mice, and lessthan 2% and 0.1% of CD11b+Gr1+ cells from wild-type and CD2-TFF2 animalswere positive for BrdU staining.

TFF2 can modulate the splenic accumulation of myeloid progenitor cells,such as the granulocyte/macrophage precursor (GMP), what give rise toCD11b+Gr1+ cells following their expansion in the spleen (A42). Therelative colony-forming capacity of splenocytes obtained from all groupsof mice on day 19 after DSS treatment was studied. Splenocytes fromCD2-TFF2 mice showed the lowest capacity to form colonies, whilesplenocytes from TFF2-null mice formed significantly more coloniescompared with those from wild type and CD2-TFF2 counterparts (FIG. 30).In parallel FACS analysis was used to evaluate the number of splenicprecursors GMP by defining them as Lin-IL-7Rα-c-kit+Sca−1− CD16/32+CD34+cells (FIG. 16B) (A43). TFF2-null mice show at least 10 fold highernumber of GMP precursors compared with wild and transgenic mice (FIG.31). In the AOM/DSS model, splenocytes from TFF2-null mice again showeda greater capacity to form colonies on granulocyte/macrophage supportingmedia and a greater number of splenic GMPs compared to splenocytes fromwild-type and CD2-TFF2 mice (FIG. 32).

FACS sorted DSS-induced CD11b+Gr1+ cells from TFF2−/− mice contained amixed population of cells with ring shaped and large nucleus segmentednuclei (FIG. 9A) that expressed macrophage antigen F4/80, dendritic cellmarker CD11c, CD31 and CD115 but low levels of MHC class II andco-stimulatory molecules CD86, CD80 and CD40 (FIG. 61A) (A44-A47). Theyalso were positive for monocytic Ly6C and granulocytic Ly6G markers witha higher proportion of the subpopulation expressing Ly6G antigen (FIG.61B). DSS-induced CD11b+Gr1+ cells sorted from the spleens of TFF2−/−mice displayed weak or no suppression on proliferation of polyclonallyactivated CD4+ T-cells in vitro.

Splenic CD11b+Gr1+ cells from tumor-bearing TFF2−/− mice highlyexpressed the CD80 co-stimulatory molecule on their surface (FIG. 36).CD80+CD11b+Gr1+ have been shown to be myeloid derived suppressor cells(MDSC) that accumulated in spleen, ascites and tumor tissue (A48), withCD80 playing a role in suppression of T-cell specific response. Sortedsplenic CD11b+Gr1+CD80+ cells significantly inhibited in vitroproliferation of CD4+ T—cells activated with CD3/CD28 antibody andshowed a decrease of INF-γ production (FIGS. 33, 34). In contrast,CD11b+Gr1+ cells from spleens of CD2-TFF2 mice that expressed much lowerlevels of surface CD80 did not suppress proliferation/INF-γ expressionof activated T-cells (FIGS. 61A, 61D-E). Addition of CD80 neutralizingCD80 Ab significantly abrogated the suppressive effect of CD11b+Gr1+cells on T-cells proliferation (FIG. 35). CD11b+Gr1+ cells did notproduce nitric oxide or arginase I.

The effect of TFF2 on the in vitro growth of sorted CD11b+Gr1+ cellsfrom spleen of TFF2−/− mice CD11b+Gr1+ cells was tested. The effect ofTFF2 on IMCS grown in presence of low concentration (5 ng/ml) of GM-CSFin medium after 7 days of culture was tested. The number of viable cellsdecreased in a dose-dependent manner upon TFF2 supplementation in range0.2 μM-4 μM (FIG. 10B). Addition of TFF2 resulted in a decrease of BrdUuptake by CD11b+Gr1+ cells in a dose-dependent manner suggesting thatTFF2 inhibited their proliferation (FIG. 37). This was associated withan 8 fold decrease of cyclin-dependent kinase D1 gene expression as hasbeen shown by microarray analysis and validated with qPCR (Table 1).Other cyclin-dependent kinases (for example E1) also were down-regulatedas much as 2-fold. Since CDK1 is master positive regulator of celldivision (A49) these in vitro studies suggest that TFF2 inhibitsproliferation of IMC largely through down-regulation of cyclin D1.Consistently, inhibition of proliferation was accompanied with anincrease in the level of negative regulators of the cell cycle such asnuclear protein 1 (Nupr1) (2.68 fold), schlafen 1 (2.16 fold), and largetumor suppressor 2 (1.96 fold). The increase of Nupr1 mRNA was validatedby q-PCR (FIG. 45). Although in most cells the increase of intracellularNupr1 is associated with the induction of apoptosis (A50) recombinantTFF2 did not induce apoptosis of CD11b+Gr1+ cells even at highconcentrations. Accordingly, microarray data expression of the CD11cmarker on CD11bG1+ cells decreased upon TFF2 treatment which isconsistent with flow cytometry results. Similarly, recombinant TFF2inhibited proliferation of CD11b+Gr1+ cells sorted from tumor-bearingmice presumably through down-regulation of cyclin D1 (FIG. 38).

TABLE 1 TFF2 regulates transcription of cell cycle genes in Gr1+CD11b+cells Symbol Description Fold change Positive regulators of cell cycleCcnd1 cyclin D1 −8.30 Ccnd2 cyclin D2 −2.23 Ccne1 cyclin E1 −2.29 Sesn3sestrin 3 −3.51 Cep55 centrosomal protein 55 −2.34 Prc1 proteinregulator of cytokinesis 1 −2.32 Spag5 sperm associated antigen 5 −2.226-Sep septin 6 −2.20 4-Sep septin 4 −2.19 Cenpe centromere protein E−2.19 Cenpf centromere protein F −2.14 Aurkb aurora kinase B −2.09 Kif23kinesin family member 23 −2.13 Negative regulators of cell cycle Nupr1nuclear protein 1 2.68 Slfn1 schlafen 1 2.16 Lats2 large tumorsuppressor 2 1.99

Recombinant TFF2 Delivered by Adenovirus Vector Suppresses ColonicCancer Progression in AOM/DSS Model.

To address whether administration of TFF2 is able to suppress cancerdevelopment in the AOM/DSS model adenovirus vectors expressing mouserecombinant TFF2 (Ad-TFF2) were generated. mTFF2 protein expression byeukaryotic cells transfected with Ad-TFF2 was validated in western blot(FIG. 50). After one dose of Ad-TFF2 injection (5×10⁸ pfu per mouse)TFF2 protein was detected in mouse blood, spleen and liver (FIG. 51).Cancer was initiated by a single injection of AOM following DSStreatment, after 1 week TFF2−/− mice were injected with adenovirusvector expressing recombinant mouse TFF2 (Ad-TFF2) and adenovirus vectoralone (Ad-Fc) as a control (FIG. 47). All mice developed tumors by 13weeks after AOM/DSS administration; however mice injected with Ad-TFF2developed statistically less number of tumors (FIG. 57). The decrease oftumor burden was associated with a lower proportion of CD11b+Gr1+ cellsin spleen and peripheral blood, but not in bone marrow (FIG. 56). TFF2was detected in the blood by western blot on day 3 after virusadministration (FIG. 54). These experiments were repeated with wild typemice with similar results.

Discussion

Gastric epithelial TFF2 can have a mucosal protective and restitutivefunction in the stomach and colon (A57-A59). Earlier studies showed thatTFF2 increased the viscosity of mucus that covers and protects theepithelium, and promoted epithelial restitution by stimulating themigration of epithelial cells to sites of injury (A6, A7, A57, A59,A60). Accumulating data suggest that TFF2 is a modulator ofgastrointestinal as well as systemic inflammation (A15, A18).

The role of trefoil peptides derived from an immune cell compartment hasnot been investigated. Splenic T-cells were identified as a source ofTFF2 under normal physiological conditions, as well as underinflammatory conditions (A61). The TFF2 level was controlled in thespleen via the cholinergic anti-inflammatory pathway and wasdown-regulated by vagotomy. TFF2 derived from T-cells contributes inamelioration of DSS-induced colitis. In a cancer model of AOM/DSS T-cellderived TFF2 plays a role in the suppression of carcinogenesis. Resultsfrom the in vivo models indicate that TFF2, in addition to its knownbarrier and reparative function in gastrointestinal tract, is alsoinvolved in an anti-tumor mechanism provided by T-cells. This anti-tumormechanism can also be provided by adenovirus delivered TFF2 in theAOM/DSS model. Transgenic mice overexpressing TFF2 under hCD2 promoterdeveloped less cancer while TFF2-null mice display highest tumor burden.Recombinant TFF2 delivered by the adenovirus system also suppressedtumor development. Importantly, suppression of tumorigenesis isassociated with a decrease in the number of CD11b+Gr1+ cells validatingtheir role in cancer progression.

Expansion of IMCs is associated with an increase in Ki67+ or BrdU+ cellswithin splenic CD11b+Gr1+ cells, suggesting their higher proliferationin the spleen of TFF2− deficient versus WT and CD2-TFF2 transgenic mice.Splenocytes from TFF2−/− mice treated with DSS form more colonies onmedium supporting granulocyte/macrophage precursors than splenocytesfrom wild type and transgenic mice. In addition, supporting the in vivoobservations, recombinant TFF2 directly suppressed CD11b+Gr1+ cellproliferation in vitro.

Splenomegaly in DSS-treated TFF2-null mice resulted from the expansionof immature myeloid cells, presumably as a consequence of extramedullaryhematopoiesis. Bone marrow is the major source of CD11b+Gr1+ cells andtheir precursors under normal and pathological conditions (A51-A53),with egress regulated through the CCR2 receptor (A54). However, recentstudies have suggested that the spleen is an important source ofextramedullary myelopoiesis under conditions of severe inflammation andcancer (A42, A43, A55, A56).

In the AOM/DSS model spleen-derived CD11b+Gr1+ cells in TFF2−/− mice areMDSCs and show profound suppression on T-cell proliferation anddecreased IFN-γ production in vitro. MDSC from the spleen oftumor-bearing TFF2−/− mice express high levels of the surface markerCD80 compared with nonsuppressive CD11b+Gr1+ cells from transgenicCD2-TFF2 mice (without visible tumor in colon) post AOM/DSS regimen. Asimilar phenotype for CD11b+Gr1+CD80+ cells with suppressive functionshas been described for melanoma patients, mouse ovarian carcinoma and4T1 mammary carcinoma models (A62, A63). CD80 expressed on CD11b+Gr1+cells ligates with CTLA-4 and transduces inhibitory signals in T-cells(A48). Consistently, the growth of ovarian 1D8 tumors was retarded inCD80-deficient mice due to a decrease of arginase I activity inCD11b+Gr1+MDSC (A64).

Since MDSCs are an important factor in promoting cancer progressionseveral strategies has been suggested in an attempt to eliminate MDSCsin vivo or suppression their activity. Such as treatments includeall-trans-retinoic acid (ATRA) (A66), 1α25-dihydroxyvitamin D (A67),administration of gemcitabine (A68), or 5-Fluorouracil (A69) to induceapoptosis, administration of phosphodiesterase-5 inhibitors in order todownregulate arginase and nitric oxide synthetase activities (A70).Indeed, two widely used anticancer cytotoxic agents, 5FU and Gem killMDSC but they also show side effects by inducing IL-1β release thatenhances IL-17 production and accelerates tumor growth (A71). Thefindings suggest that TFF2 is able to suppress expansion of myeloidcells and delivering recombinant TFF2 in the blood of a subjectrepresents a new strategy to control MDSCs population even underconditions promoting cancer development.

TABLE 2 Brief Summary of Microarray Dat. GeneChip ® Mouse genome 430A2.0 Array Total analyzed genes: ~14,000 (22,600 probes) with changedmRNA expression 5,810 (P < 0.05) upon TFF2 treatment: (>2 fold) 807 (>3fold) 309 (>4 fold) 188

TABLE 3 22 genes upregulated (>10 fold) by TFF2 in Gr1CD11b cells FoldSymbol Description Change Up Apoe apolipoprotein E 89.8 Mcpt8 mast cellprotease 8 72.5 Cxcl5 chemokine (C—X—C motif) ligand 5 62.8 Cpa3carboxypeptidase A3, mast cell 54.7 Il12a interleukin 12a 33.1 F2rcoagulation factor II (thrombin) receptor 30.8 Ctsg cathepsin G 20.9FeerIa Fc receptor, IgE, high affinity I, alpha 19.9 polypeptide Prtn3proteinase 3 18.2 Mpo myeloperoxidase 17.4 Lipg lipase, endothelial 16.8Epx eosinophil peroxidase 15.5 Ms4a3 membrane-spanning 4-domains, 15.3subfamily A, member 3 Cyp11a1 cytochrome P450, family 11, 14.5 subfamilya, polypeptide 1 Fst follistatin 14.1 Akr1c18 aldo-keto reductase family1, member C18 13.0 My110 myosin, light chain 10, regulatory 11.9 Saa3serum amyloid A 3 11.8 Ptgs2 prostaglandin-endoperoxide synthase 2 11.5Ceacam10 carcinoembryonic antigen-related cell adhesion molecule 10 10.6Elane elastase, neutrophil expressed 10.0 Cd55 CD55 antigen 10.0

Key: Secreted Cytosolic Membrane

TABLE 4 11 genes downregulated (>10 fold) by TFF2 in Gr1+CD11b+ cellsFold Change Symbol Description Down Ly86 lymphocyte antigen 86 −19.7K1rb1b killer cell lectin-like receptor subfamily B −19.3 member 1BHepacam2 HEPACAM family member 2 −16.0 Cldn1 claudin 1 −15.6 Mfge8 milkfat globule-EGF factor 8 protein −14.1 Ciita class II transactivator−14.0 Il2ra interleukin 2 receptor, alpha chain −12.0 Axl AXL receptortyrosine kinase −10.9 Zbtb46 zinc finger and BTB domain containing 46−10.7 Rtn1 reticulon 1 −10.6 Ccl17 chemokine (C-C motif) ligand 17 −10.1

Key: Secreted Nuclear Membrane

TABLE 5 TFF2 effect on integrin expression in CD11bGr1 cells SymbolDescription Component of: Fold Change Itgax integrin alpha X CD11c -2.3*Itgam integrin alpha M CD11b 1.3 Itga4 integrin alpha 4/CD49d VLA-4 1.2Itga4 integrin alpha 4/CD49d VLA-4 1.1 Itga4 integrin alpha 4/CD49dVLA-4 1.0 Itgav integrin alpha V VNR 1.3 Itgav integrin alpha V VNR 1.3Itgav integrin alpha V VNR 1.1 Itgb21 integrin beta 2-like Homodimer8.8** Itgb2 integrin beta 2/CD18 CD11c/Cd11b 1.1 *= Detected by FC **=Expression in mouse neutrophils only, no orthologs

Summary: Adenoviral delivery of TFF2 suppresses colon cancer in responseto AOM/DSS.

Conclusion: These results show that adenoviral delivery of mTFF2expression can suppress gastrointestinal tumorigenesis through reducingthe proliferation of IMCs.

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Example 7 Tumor-associated MDSC

The role of TFF2-mediated suppression of MDSCs in cancer development canalso be tested in mouse models of mammary cancer (for example, seeHennighausen (2000) Breast Cancer Res. 2(1): 2-7; and Fantozzi et al.,(2006) Breast Cancer Res. 2006; 8(4): 212, each of which are herebyincorporated by reference in their entireties). A large number of wellvalidated genetically engineered models (GEM) of breast cancer have beendeveloped over the past few decades, and many (e.g. MMTV-HER2/Neu orMMTV-Wnt-1 or MMTV-PyV-mT) are widely available. In addition, a H.hepaticus infection model has recently been utilized to stimulate breastcancer development in Apc/Min mice (Rao V P et al, Cancer Res 2006;66:7395-400).

The role of TFF2-mediated suppression of MDSCs in cancer development canalso be tested in mouse models of prostate cancer (for example, see Jeetet al (2010) Cancer Metastasis Rev. 29(1):123-42; Zhou et al., (2010) JAndrol. 31(3):235-43; Ahmad et al., (2008) Expert Rev Mol Med. 10:e16;Havens et al., (2008) Neoplasia. 10(4): 371-379; Valkenburg and Williams(2011) Prostate Cancer, Volume 2011, Article ID 895238,doi:10.1155/2011/895238, each of which are hereby incorporated byreference in their entireties). A large number of well validatedgenetically engineered models of prostate cancer have been developedover the past few decades, and many are widely available.

In addition, the role of TFF2-mediated suppression of MDSCs in cancerdevelopment can also be tested in mouse models of lung cancer (forexample, see Meuwissen and Berns (2005) GENES & DEVELOPMENT 19:643-664;Kwon and Berns (2013) Molecular Oncology 7(2):165-177; de Serrano andMeuwissen (2010) Eur Respir J. 35: 426-443, each of which are herebyincorporated by reference in their entireties). A large number of wellvalidated genetically engineered models of lung cancer have beendeveloped over the past few decades, and many are widely available.

Using one or more of these mouse models, the ability of TFF2 to suppressmyeloid progenitors and cancer initiation and progression can be tested.To start with, the CD2-TFF2 transgenic mice can be crossed to a breastcancer mouse model (non-limiting examples include: MMTV-HER2/Neu orMMTV-Wnt-1 or MMTV-PyV-mT), and the development of breast cancer can befollowed over time. To study prostate cancer, the CD2-TFF2 transgenicmice can be crossed to a prostate cancer mouse model (non-limitingexamples include: Androgen Receptor Knockout mouse,PB-Cre4×PTEN(loxP/loxP) mouse, TRAMP (for transgenic adenocarcinomamouse prostate), FG-Tag mouse, PB-Neu, and LADY), and the development ofprostate cancer can be followed over time. To study lung cancer, theCD2-TFF2 transgenic mice can be crossed to a lung cancer mouse model(non-limiting examples include: CC10-Tag/CC10-hASH1, K5-E6/E7,CCRP-H-Ras, and MMTV-TGF-β1 DN), and the development of lung cancer canbe followed over time.

In addition, the use of adenoviral-TFF2 delivery to these animals can beused, through tail vein injection of the recombinant virus. Endpointscan include tumor number, tumor size, and tumor load. This can becorrelated with the level of circulating MDSCs and the percentage ofMDSCs in the mouse spleen and bone marrow. Without being bound bytheory, TFF2 will reduce MDSC expansion and cancer development in eitherthe breast, prostate, or lung.

Example 8 TFF2 is Upregulated in Memory T Cells Through AdrenergicStimulation

The studies described herein suggested that the vagus nerve upregulatesTFF2 expression in CD4+ memory T cells through an adrenergic (e.g.noradrenaline) pathway. This conclusion was based on previous studies onthe inflammatory reflex, which documented that the vagus stimulatedsplenic nerves, which released noradrenaline to stimulate CD4+ memory Tcells. These T cells in turn produce acetylcholine, which inhibitcytokine production (TNF-alpha) by macrophages. However, in order todemonstrate that adrenergic signaling also regulates the production ofTFF2 by T cells, studies were carried out with isoproterenol stimulationof memory T cells. These studies (FIG. 62) showed that TFF2 mRNAexpression was tightly regulated by adrenergic stimulation with a15-fold increase seen with 500 nM of isoproterenol.

Example 9 T Cells are the Primary Source of TFF2 in the HematopoieticSystem

The studies described herein supported the notion that memory CD4+ Tcells are the major source of TFF2 mRNA expression in the spleen. Theyappear to be the same T cells as those targeted by the vagus nerve,which have been reported to be positive for Chat expression.

To further address this issue, a TFF2-EGFP BAC transgenic mouse wasgenerated. It was confirmed that the transgene was expressed in thestomach, pancreas and spleen. Splenic memory T cells were sorted usingCD4+CD44hiCD62Llo for flow sorting, and it was confirmed that thesecells strongly expressed the TFF2-EGFP transgene, while the rest of thesplenic T cells did not (FIG. 63A).

In addition, mouse splenocytes were treated with PMA/ionomycin, whichcombines cell stimulation with protein transport inhibition, and thenFACS sorted memory T cells (CD4+CD44hiCD62Llo), then fixed,permeabilized and performed immunofluorescent staining (FIG. 63B). Thesestudies confirmed colocalizaiton of TFF2 and CD44.

Example 10 T Cells are the Main Source of TFF2 in the Spleen

TFF2 is expressed in most mammals primarily in the gastric mucosa, butas described herein is also in low levels in the spleen. The datadescribed herein suggested that T cells are the major source of TFF2within the spleen. However, in order to confirm this finding, varioushematopoietic populations in the spleen were sorted, and then assessedTFF2 expression by RT-PCR (FIG. 64). These studies showed that TFF2 ismost strongly expressed in the CD4+ T cell population, with littleexpression in other compartments.

Example 11 Decreased Tumor Counts in TFF2-Overexpressing Mice are Due toT Cell Activation

The data described herein showed that TFF2 overexpression inhibitsexpansion of myeloid-derived suppressor cells (MDSCs), which wasassociated with an increase in CD8+ T cells in the colon and a decreasein colonic tumors. To show that the decreased tumors in the colon aredue to increased infiltration with activated T cells studies werecarried out to show that CD8+ T cells are essential for the decreasedcolonic tumors seen in TFF2 transgenic mice (FIG. 65). CD2-TFF2transgenic mice were treated with AOM/DSS and then treated with ananti-CD8 antibody that depleted CD8+ T cells in the mouse. The antibodywas given twice a week for 12 weeks, with an isotype control. Thisdepletion of CD8+ T cells resulted in a marked increase in tumor numberand tumor load compared to the control, demonstrating the CD8+ T cellsare necessary for the tumor inhibitory effect of TFF2.

Example 12 TFF2 Leads to Activated CD8+ T Cells in CD2-TFF2 TransgenicMice

In order to show that the TFF2-overexpressing mice have activated CD8+ Tcells, CD8+ T cells were sorted from the spleens of transgenic mice, andthe amount of interferon-gamma and granzyme B (markers of activated Tcells) was measured by ELISASPOT (FIG. 66). These studies showed thatCD2-TFF2 transgenic mice had significantly increased IFN-g and Grb intheir splenic CD8+ T cells compared to TFF2 null mice, followingAOM+DSS.

Example 13 Myeloid Cells Instructed in a TFF2-Deficient Spleen SuppressCD8 T Cells and Promote Colon Cancer

As described herein, TFF2 overexpression, whether via the CD2-TFF2transgene or through Ad-TFF2 overexpression, suppresses MDSCs andsuppresses colon tumors. To show that the suppression of colon tumorswas through MDSCs, or that MDSCs in TFF2 deficient mice induced coloncancer, adoptive transfer of the MDSCs from TFF2−/− mice treated withAOM/DSS into CD2-TFF2 mice that have been treated with AOM/DSS wascarried out. This addressed whether MDSCs from TFF2−/− mice that areprone to cancer can change the cancer-resistant CD2-TFF2 mice to micethat are susceptible to cancer. Is the absence of these MDSCs,instructed in the spleen of mice lacking TFF2, the critical factormissing in the process of cancer initiation by AOM/DSS. FIGS. 67A-Bshows MDSCs contribute to carcinogenesis in AOM/DSS-induced colon cancermodel.

Example 14 Splenic MDSC from TFF2-Null Mice Promote ColonicTumorigenesis to a Greater Extent than MDSC from CD2-TFF2 TransgenicMice

Here it was shown the direct suppression of MDSC tumor initiatingfunction by TFF2 overexpression. Myeloid precursors instructed in thespleen in the absence of TFF2 can migrate to the periphery to suppressCD8+ T cell responses, and such instruction can be altered by expressionof TFF2 in the spleen. FIGS. 68A-B shows splenic IMC from Tff2-null miceshow higher contribution in tumorigenesis vs. splenic IMC from CD2-Tff2mice.

1-51. (canceled)
 52. A method of treating a disease or condition in asubject comprising administering to the subject a TFF1 molecule, a TFF2molecule, a TFF3 molecule, or a combination thereof, wherein thetreatment is selected from the group consisting of treating a digestivesystem cancer, decreasing myeloid-derived suppressor cell proliferation,decreasing tumor growth, and treating dysplasia of the digestive system.53. The method of claim 52, wherein the TFF1 molecule, TFF2 molecule, orTFF3 molecule is a nucleic acid.
 54. The method of claim 53, wherein thenucleic acid is delivered as a viral vector.
 55. The method of claim 53,wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:
 6. 56.The method of claim 52, wherein the digestive system cancer is selectedfrom the group consisting of small intestine cancer, large intestinecancer, colon cancer, rectal cancer, and anal cancer.
 57. The method ofclaim 52, wherein the myeloid-derived suppressor cell is a tumorassociated myeloid-derived suppressor cell.
 58. The method of claim 52,wherein the myeloid-derived suppressor cell expresses a MDSC-specificsurface marker.
 59. The method of claim 52, wherein the myeloid derivedsuppressor cell does not express a MDSC-specific surface marker.
 60. Themethod of claim 58, wherein the MDSC-specific surface marker is selectedfrom the group consisting of (1) Grl1, CD11b, or a combination thereof;and (2) CD14, CD15, CD33, or a combination thereof.
 61. The method ofclaim 59, wherein the MDSC-specific surface marker is HLA-DR.
 62. A kitfor use for the method of claim 52, the kit comprising a TFF1 molecule,a TFF2 molecule, a TFF3 molecule, or a combination thereof, andinstructions of use.
 63. A method of determining the presence of, orpredisposition to, a cancer of the digestive system in a sample from asubject, the method comprising: (a) detecting the presence, absence orreduction of a TFF1 molecule, TFF2 molecule, or a TFF3 molecule in thesample, wherein absence, or reduction of the molecule indicates thepresence of, or predisposition to, a cancer of the digestive system. 64.The method of claim 63, further comprising (b) administering a TFF1molecule, a TFF2 molecule, a TFF3 molecule, or a combination thereof tothe subject where a TFF1 molecule, a TFF2 molecule, or a TFF3 moleculewas not detected.
 65. The method of claim 63, further comprisingincubating the sample with an agent that binds a TFF1 molecule, a TFF2molecule, or a TFF3 molecule, or fragment thereof.
 66. The method ofclaim 65, wherein the agent is an antibody to a TFF1 molecule, a TFF2molecule, or a TFF3 molecule.
 67. The method of claim 63, wherein thedigestive system cancer is selected from the group consisting of smallintestine cancer, large intestine cancer, colon cancer, rectal cancer,and anal cancer.
 68. The method of claim 63, wherein the TFF1 molecule,TFF2 molecule, or TFF3 molecule is a nucleic acid.
 69. The method ofclaim 68, wherein the nucleic acid is delivered as a viral vector.
 70. Adiagnostic kit for determining the presence of, or predisposition to, acancer of the digestive system, the kit comprising an agent that bindsto a TFF1 molecule, a TFF2 molecule, or a TFF3 molecule, andinstructions for use.
 71. The kit of claim 70, wherein the agent is anantibody to a TFF1 molecule, a TFF2 molecule, or a TFF3 molecule.